Method and apparatus for the production of soluble mhc antigens and uses thereof

ABSTRACT

The field of the invention relates in general to at least one method and apparatus for the production of soluble MHC antigens and more particularly, but not by way of limitation, to at least one method and apparatus for the production of soluble Class I and II HLA molecules. The field of the invention also includes such produced soluble Class I and II HLA molecules and their use. According to the methodology of the present invention, the soluble Class I and II HLA molecules can be produced from either gDNA or cDNA starting material.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is continuation of U.S. Ser. No. 11/099,283,filed Apr. 4, 2005; which is a continuation of U.S. Ser. No. 10/022,066,filed Dec. 18, 2001, entitled “METHOD AND APPARATUS FOR THE PRODUCTIONOF SOLUBLE MHC ANTIGENS AND USES THEREOF,” now abandoned. Theapplication U.S. Ser. No. 10/022,066 claims priority under 35 U.S.C. §119(e) of provisional U.S. Ser. No. 60/256,410, filed Dec. 18, 2000,entitled “HLA PRODUCTION FROM GENOMIC DNA”; provisional U.S. Ser. No.60/256,409, filed Dec. 18, 2000, entitled “HLA PROTEIN PRODUCTION FROMcDNA”; and provisional U.S. Ser. No. 60/327,907, filed Oct. 9, 2001,entitled “PRODUCTION OF SOLUBLE HUMAN HLA CLASS I PROTEINS FROM GENOMICDNA,” the contents of all of which are hereby expressly incorporated intheir entirety by reference.

The application U.S. Ser. No. 10/022,066 is also a continuation-in-partof U.S. Ser. No. 09/465,321, filed Dec. 17, 1999, entitled “METHOD ANDAPPARATUS FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS,” now abandoned,the contents of which are hereby expressly incorporated in theirentirety by reference.

The application U.S. Ser. No. 10/022,066 is also continuation-in-part ofU.S. Ser. No. 09/974,366, filed Oct. 10, 2001, entitled “COMPARATIVELIGAND MAPPING FROM MHC POSITIVE CELLS,” the contents of which arehereby expressly incorporated in their entirety by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

At least a portion of the invention was developed under funding from theNational Institute of Health (“NIH”) under contract Nos. No1-A1-45243and No1-A1-95360. As such, the Government may own certain rights in andto this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the invention relates in general to at least one method andapparatus for the production of soluble MHC antigens and moreparticularly, but not by way of limitation, to at least one method andapparatus for the production of soluble Class I and II HLA molecules(i.e., sHLA). The field of the invention also includes such producedsoluble Class I and II HLA molecules and their uses. According to themethodology of the present invention, the soluble Class I and II HLAmolecules can be produced from either gDNA or cDNA starting material.One such exemplary, but non-limiting, use is the formation of atetrameric sHLA (or other multimeric complex) complex which may be usedto test immunogenicity of peptide ligands of interest—i.e., will apeptide ligand of interest provoke a CTL response and/or preferentiallybind a CTL.

2. Brief Description of the Background Art

Class I HLA molecules are polymorphic human glycoproteins thatendogenously bind and then extracellularly present peptide ligands toCD8⁺ T lymphocytes. Polymorphisms within the class I peptide bindinggroove are positioned to moderate ligand binding and presentation tosuch immune system cells. To date, the small quantities of naturalligands available to those of skill in the art has limited theunderstanding of precisely how polymorphism alters peptide binding andin turn, vaccine development and, more basically, if a peptide ligand ofinterest will provoke a CTL mediated immune response. In order toaddress the impact of polymorphism upon antigen presentation, theinventors developed the novel approach disclosed herein—i.e., thatligand presentation overlaps exist across the polymorphisms and thatthese overlaps distinguish divergent class I peptide binding grooves.Utilizing this novel approach and coupling it with a unique hollow-fibercell culture scheme and utilizing a mass spectrometric ligand mappingapproach, large quantities of peptides eluted from soluble class I andclass II molecules can be obtained for detailed analyses, vaccinedevelopment, and functional testing.

Initially, peptide ligands were extracted from five different HLA-B15allotypes and subsequently examined. Mapping and characterizing theligands obtained from these allotypes demonstrated that they: (i) varyin length from 7 to 12 residues; (ii) are more conserved at their Ctermini than at their N-proximal residues; and (iii) are presented asoverlaps contingent on C-terminal preferences. These results provideinsight into class I and class II ligand loading not available via othermethods, demonstrating that an elemental role is played by a peptideligand's C terminus during endogenous binding and provides the startingmaterial for a multimeric complex to be used to test the functionalityof a peptide ligand of interest. The data obtained, and disclosedherein, validates, and illustrates the unique methods disclosed hereinfor the production of sHLA from either gDNA or DNA starting material andthe uses to which this sHLA material may be put.

Class I and class II MHC molecules, designated HLA class I and class II,respectively, in humans, bind and display peptide antigens upon the cellsurface. The peptides they present are derived from either normalendogenous proteins (“self”) or foreign proteins (“nonself”). Nonselfproteins include items such as the products of malignant transformationor intracellular pathogens. In this manner, class I and class IImolecules convey information regarding the internal fitness of a cell toCD8⁺ CTLs which are activated upon interaction with “nonself” peptides.Such activation may lead the CD8+ CTLs to kill and/or suppress a cellwhich is malignant or contains intracellular pathogens.

Examination of HLA by serologic and molecular methods by those ofordinary skill in the art continues to demonstrate that the class IHLA-A, B, and C molecules are encoded by the most polymorphic genes inmammals. Translating class I polymorphism into the tertiary structure ofthe class I molecule indicates that residues positioned to affect classI peptide presentation to T lymphocytes are most frequently affected bythe mutagenic events which diversify class I loci. Throughout the world,HLA class I molecules exhibit a high degree of polymorphism that isgenerated by systematic recombinatorial events and collectively allowsfor the presentation of a vast array of different peptides. Dependingupon allelic composition, two individuals' molecules may not necessarilybind the same peptides with equal affinity or even at all.

While the general structure and function of MHC class I molecules hasbeen reasonably well studied and established, their polymorphic natureand how they specifically influence the capacity of class I in peptidebinding and presentation remains an issue of persistent inquiry by thoseof ordinary skill in the art. The nature of precise overlaps in peptidebinding specificity to HLA class I is particularly ill-defined at thecurrent time due to the complexity of peptides bound. For example, thisand other issues must be clarified in order to effectively pursuevaccines capable of eliciting protective CTL responses across anextensive population range. Unraveling the functional significance ofclass I polymorphism is an important issue that requires anunderstanding of how the mutagenic events diversifying the class Ibinding groove differentially moderate the presentation of peptideligands.

The heavy chains of class I molecules are encoded within the MHC and,upon assembling into heterodimers with the light chain, β₂m, areresponsible for selectively gathering endogenously processed peptides.Once peptides are collected, mature class I molecules transport thebound peptides to the cell surface where receptors on CD8⁺ T lymphocytesengage the class I molecules to inspect the ligands. CTLs may then betriggered by class I molecules bearing virus or tumor-derived peptides.

With respect to the background art, hereafter numerous references aredisclosed which detail one or more aspects of the background art as itrelates to the novel methods and uses of the present invention. As such,each reference listed should be understood as being wholly incorporatedby reference herein in their entirety as though the reference were fullytranscribed herein. In this manner, one of skill in the art given thepresent specification would be fully informed and could truly appreciatethe novel and unprecedented nature of the invention(s) disclosed andtaught herein. The full and complete citations for each reference areappended hereto after the detailed description and before the claims.

The class I molecules expressed upon the nucleated cells of allvertebrate systems studied to date (for example, Ennis et al. 1988;Winkler et al. 1989; Kaufman et al. 1990; Lawlor et al. 1990; Trowsdale1995; Prilliman et al. 1996; Antao et al. 1999) are heterodimerscomposed of a glycosylated 45 kDa heavy chain (α-chain) and a 12 kDalight chain (β₂m). In humans, heavy chains are encoded at 3 loci (B, C,and A) within the MHC on the short arm of chromosome 6 (FIG. 1A). FIG.1B illustrates each α-chain comprised of α₁, α₂, and α₃ domains, as wellas a transmembrane domain, which tethers the molecule to the cellsurface and a short C-terminal cytoplasmic domain (Björkman and class Ilocation and heavy chain coding region).

X-ray crystallography (Björkman et al. 1987a; Madden et al. 1991; Saperet al. 1991; Madden et al. 1992; Collins et al. 1995; Reid et al. 1996;Smith et al. 1996a; Smith et al. 1996b; Glithero et al. 1999) hasillustrated details of the structural relationship of the extracellularα-chain domains and β₂m (FIG. 2). The membrane-proximal domains, α₃ andβ₂m, associate in an immunoglobulin fold structure. The membrane-distalα₁ and α₂ domains together create a closed basket-like structure thatsits atop the α₃ and β₂m structure (FIG. 2A). It consists of twoα-helical “walls” with a “floor” created by eight anti-parallel β-sheets(Björkman et al. 1987a; Madden 1995; and FIG. 2, B and C). In theearliest studies, detection of electron density situated in the α₁/α₂groove helped to clarify the experimentally-suspected occupancy ofpeptide fragments 8-10 residues long and thus the function of class Imolecules in presenting such peptides upon the cell surface (Björkman etal. 1987b). Initial crystallographic studies designated six subsites,A-F (FIG. 3), or “specificity determining pockets,” that constitute thepeptide binding groove (Garrett et al. 1989; Saper et al. 1991; Fremontet al. 1992; Matsumura et al. 1992; Elliott et al. 1993; Young et al.1995). In addition to crystal structure analyses, thermodynamicstability studies indicate that networks of hydrogen bonds to structuralresidues lining the A- and F-pockets, which lie at opposite ends of thegroove, serve to fasten a peptide by its N and C termini, respectively(Bouvier and Wiley 1994); these two pockets are thus implicated in thefixed orientation of a peptide within the binding groove.

Class I molecules primarily associate with peptide fragments, thusforming α-chain/β₂ m/peptide trimolecular complexes, via an endogenousprocessing pathway during their assembly (Germain 1994; Heemels andPloegh 1995; Lehner and Cresswell 1996; York and Rock 1996; Pamer andCresswell 1998); in fact, the very binding of peptides is essential forthe stabilization and expression of these molecules (Ljunggren et al.1990; Townsend et al. 1990; Elliott 1991). The class I α-chain and β₂mare cotranslationally translocated into the ER lumen (Townsend et al.1990; Germain and Margulies 1993; Neefjes et al. 1993), where theα-chain remains anchored to the ER membrane and is stabilized prior toassociation with β₂m and/or peptide through interactions with variouschaperone proteins, including BiP, ER-60, calnexin, calreticulin, andtapasin (Nöβner and Parham 1995; Sadasivan et al. 1996; Suh et al. 1996;Spee and Neefjes 1997; Harris et al. 1998; Lindquist et al. 1998).Although several alternative proteolytic processing and transportpathways, certainly exist (for example, Hsu et al. 1991; Henderson etal. 1992; Kozlowski et al. 1992; Roelse et al. 1994; Snyder et al. 1994;Ferris et al. 1996; Craiu et al. 1997; Luckey et al. 1998; Mosse et al.1998; Wang et al. 1998; Young et al. 1998), it is believed that themajority of peptides the nascent class I molecules interact with aredelivered to the ER in a distinct series of events.

Proteins in the cytoplasmic compartment are first enzymatically degradedinto peptides of relatively uniform length by an ATP-dependentproteasome complex (Coux et al. 1996). Some proteasome componentsinclude the IFN-γ inducible subunits LMP2 and LMP7; these are themselvesencoded within the MHC (Gaczynska et al. 1994). The typically nonamericfragments produced are then actively conveyed across the ER membrane viaa dimer of TAP1/TAP2, an MHC-encoded ATP-binding cassette transporter(Monaco et al. 1990; Parham 1990; Grandea III et al. 1995). Once insidethe ER, a processed peptide can be captured within the α₁/α₂ groove of aclass I molecule and a stable trimer is formed (Germain and Margulies1993; Smith et al. 1995). This trimeric α-chain/β₂ m/peptide complex isthen transported through the Golgi complex and ultimately to theextracellular surface. The processes of class I assembly and transportare summarized in FIG. 4.

Following cell surface localization, mature complexes of class I bearingpeptide antigens become available for interaction with receptors onmonocytes, B and T lymphocytes, and NK cells (Townsend and Bodmer 1989;Yokoyama 1993; Lanier and Phillips 1996; Borges et al. 1997; Cosman etal. 1997). Their primary, and to date most thoroughly examined, naturalreceptor appears to be the TCR of T lymphocytes bearing the CD8heterodimer. Site-directed mutagenesis and crystallographic studiesindicate that the Vα and V_(β) domains of heterodimeric TCRs associatein a diagonal fashion across the top surface of the structure formed byMHC α₁ and α₂ (Hogan et al. 1988; Lombardi et al. 1991; Moots 1993;Tussey et al. 1994; Garboczi et al. 1996; Garcia et al. 1996; Parham1996; Björkman 1997; Smith and Lautz 1997; Manning et al. 1998). Thehypervariable CDRs of V_(α)/V_(β) contact specific regions of thisinterface (FIG. 5). Both precursor and effector CTLs are defined asbeing class I-restricted in that they are only capable of recognizingand responding to antigens displayed in the context of these molecules(Zinkernagel and Doherty 1974).

Since the antigens presented to CD8⁺ T lymphocytes are predominantlyobtained through the processing of intracellular proteins as previouslydescribed, class I molecules figuratively serve as external banners thatadvertise the inner contents of the cells. Indeed, these antigens ofpeptide ligands indicate to the immune system as a whole which cells areto be eliminated and/or protected. Malignancies and/or pathogenseffectively use this system to camouflage their existence and therebyescape detection and elimination by CD8+ CTLs, for example.

Thymic education of lymphocytes prevents activation in response tocharacteristic cell-derived peptides (Robey and Fowlkes 1994), butpeptides acquired through the degradation of atypical proteins arerecognized and induce cytolysis (Townsend et al. 1985; Gotch et al.1988; Walker et al. 1988; Clark et al. 1995). Therefore, it is notsurprising that CD8⁺ T lymphocytes play a critical role in controllingand/or eliminating infected and neoplastic cells.

CD8+ T lymphocytes are implicated in immunity to pathogens such asviruses, which are intracellular invaders that utilize the host cell'sbiosynthetic machinery to produce their own foreign proteins (Yap et al.1978; Lin and Askonas 1981; Jamieson et al. 1987; Harty and Bevan 1992;Riddell et al. 1992; Kulkarni et al. 1995; Heslop et al. 1996; Schmitzet al. 1999). CTL responses are likewise extended to include stimulationby aberrant proteins such as those associated with malignancy (Vose andBonnard 1982; Muul et al. 1987; Coulie et al. 1992; Melief 1992;Kittlesen et al. 1998; Shichijo et al. 1998). In fact, class I moleculesare capable of binding and presenting to CTLs any protein introducedinto the endogenous processing pathway by either natural or artificialmeans (Gooding and O'Connell 1983; Moore et al. 1988; Yewdell andBennink 1990; Bertoletti et al. 1991; Donnelly et al. 1993; Ikonomidiset al. 1994; Ballard et al. 1996; Day et al. 1997; Goletz et al. 1997;Kim et al. 1997). This knowledge serves as a motivating factor behindthe development of both protein/peptide-based vaccines and othertherapeutics intended to elicit protective CTL responses to microbialpathogens and other abnormalities, which otherwise remaincytoplasmically concealed from detection.

Fully understanding the role of class I molecules in ligand presentationas described above is complicated by α-chain polymorphism. Class Istructural differences resulting from genetic variation confer extremeheterogeneity upon regions of the molecule that interact with peptides.The knowledge of how polymorphism specifically impacts the naturalpresentation of peptide epitopes upon the cell surface is consequentlylimited.

Class I MHC polymorphism was first documented in mice (Gorer 1936; Gorer1937; Nathenson et al. 1981) and next studied in humans by serology(Dausset 1958; Payne and Hackel 1961; van Rood 1969); however, firstprotein and then DNA sequencing studies precisely demonstrated thisgenetic variability to be most concentrated throughout the exons of theα₁ and α₂ heavy chain domains at positions affecting amino acid residuesthat line the walls and floor of the previously described peptidebinding groove (Orr et al. 1979; Tragardh et al. 1979; Rojos et al.1987; Parham et al. 1988; Parham et al. 1989; Parham et al. 1995). It isthe more centrally-located binding pockets (B-E), together with specificresidues within the F-pocket, that appear to be the most polymorphic(Chelvanayagam 1996; Kostyu et al. 1997). Changes in the physicochemicalproperties of amino acid side chains within the groove can influence thestability with which given peptides interact during the assembly ofα-chain/β₂ m/peptide trimers (Matsui et al. 1993; Rohren et al. 1993;Salter 1994; Young et al. 1995). Therefore, despite the overallstructural conservation illustrated among class I α-chains (Björkman andParham 1990; Madden 1995), their peptide binding grooves can varydrastically from one allelic form to another; as a result variousisoforms are capable of associating with distinct arrays of peptides(Elliott et al. 1993; Smith et al. 1995; Smith et al. 1996b).

The characteristic polymorphism observed among class I molecules isthought to originate primarily through recombination and gene conversion(Kuhner et al. 1991; Parham et al. 1995); point mutations are believedto contribute more rarely to the pool of new alleles continually arising(Parham et al. 1989). Individuals inherit a set of three class I genesfrom each parent, and since their expression is codominant, a singleperson may therefore display up to six different HLA class I moleculesupon his or her nucleated cells. From these alleles, new forms evolvingprogressively within populations can be passed on to subsequentgenerations and likewise serve as templates upon which yet furtherdiversity may be introduced. This occurs through events such as singleor double recombination (Parham et al. 1988) or nonreciprocal exchangesbetween cis-oriented gene segments during gene conversion (Parham 1992).Serological cross-reactivity studies, as well as locus-specific PCRamplification and sequencing analyses, have verified the existence ofallelic subtypes, or allotypes, of closely related alleles that appearto have arisen from a common ancestral template by these molecularmechanisms (for example, Payne et al. 1978; Ooba et al. 1989; Hildebrandet al. 1994; Prilliman et al. 1996). While both inter- and intra-locusgenetic events may give rise to polymorphism, the latter is mostcommonly observed; alleles at a locus generally tend to be more closelyrelated to one another than to those present at other loci (Parham etal. 1988; Parham et al. 1995). The forces driving HLA class Ipolymorphism are believed to be those of overdominant or balancingselection (Hughes and Nei 1988; Hughes and Yeager 1998); this is basedupon values of d_(N)>d_(S) within the coding regions of α₁ and α₂ forspecificity determining pocket residues positioned to interact with thepeptide binding groove, while the contrary (d_(S)>d_(N)) is observedamong the remaining α₁/α₂ residues.

Considering the manner by which class I structural polymorphisms aregenerated and maintained demonstrates that HLA genetic variabilityaffords both advantages and disadvantages. It is beneficial in ensuringthat at least a small portion of the human population will possess classI molecules capable of: (i) binding immunogenic peptides derived fromany given pathogen; (ii) presenting those peptides to CTLs; and (iii)evoking a protective immune response. In short, annihilation of thespecies is guarded against by molecular diversity (Parham 1992). Theconcept of heterozygote advantage through polymorphism as a mechanismfor effectively allowing broader peptide binding abilities and thusbroader CTL recognition of pathogenic peptides has been stronglyemphasized from a statistical perspective (Hughes and Nei 1988; Nei andHughes 1992). For example, HLA heterozygosity has been correlated withdiminished progression to disease following HIV infection (Carrington etal. 1999). In addition, at the level of individual allotypes the“nonrandomness” with which certain polymorphisms are maintained withinpopulations following their evolution supports positive naturalselection. This might occur in response to specific pathogenicpressures, as seen in the strong association of the West African alleleHLA-B*5301 with resistance to malaria (Hill et al. 1991; Hill et al.1992), a parasitic illness endemic to West Africa. As mentionedpreviously however, the polymorphic nature of class I molecules resultsin divergent allotypes binding and presenting different peptides. CTLsthus focus on distinct portions of any given pathogen from oneindividual to another. Therefore, dissecting disease susceptibilitiesand resistance requires a grasp of how binding groove-localized aminoacid variations specifically alter ligand presentation.

Numerous previous research endeavors have been directed towardunderstanding the structural and functional nature of peptides bound byHLA complexes; though some progress has been made in analyzing themanner that peptide binding is specifically influenced through α₁/α₂substitutions, this knowledge remains limited and sometimesinconsistent. The full extent that polymorphisms dictate the degrees ofligand binding ability, stringency, and/or degeneracy (and subsequentlycell surface presentation) has, as a result, not been adequatelyresolved. Similarly, the occurrence of overlapping ligands, or identicalpeptides presented across the binding groove polymorphisms of multipledistinct allotypes, remains to be explored.

Ideally, characterizing functional overlaps would provide an advantagenot only to explore the general effects of binding groove architecturebut more specifically to understand the similarities and/or differencesof what is presented to CTLs by the class I molecules of geneticallydiverse individuals. In the search for answers to ligand bindinginfluences by α-chain polymorphisms, methods including pooled Edmansequencing, mass spectrometric analysis, and binding/reconstitutionassays have been employed. However, each approach bears its ownstrengths and limitations and none so far has been significantlysuccessful in comparatively evaluating levels of functional overlapacross class I polymorphisms. The importance of understanding peptideassociations with polymorphic class I molecules at a level of complexitynot necessarily afforded by the currently-defined strategies is thusunderscored: epitope predictions based upon methods that fail toaccurately assign possibilities for natural binding groove occupancy byeither aberrant or low-abundance peptides of varying binding affinitiesinterfered with detecting presentation overlaps among various HLA classI allotypes.

Early investigations of class I peptide ligands focused on simplifyingthe effects of polymorphism through establishing “motifs” (Rötzschke andFalk 1991; Rammensee et al. 1993; Engelhard 1994). Motifs have typicallybeen established by purifying surface-expressed class I molecules fromdetergent lysates of either transformed cells or transfectants andextracting the bound ligands with either TFA or acetic acid. Theresulting peptide pools are then subjected to consecutive cycles ofN-terminal Edman degradation (Falk et al. 1991; Jardetzky et al. 1991).

The resultant motifs from N-Terminal Edman degradation are invariablynine amino acids in length and describe, as based upon relative yieldincreases per cycle, conserved “anchor” residues, or sites ofstereochemical preferences, for peptides that are bound by a class Imolecule. These anchors, typically appearing to involve both P2 and P9of the ligands, are considered to be allele-specific and thus commonamong nearly all of the peptides bound by a given class I allotype. Theremaining positions demonstrate no overriding amino acid preferences,although the motifs of a few molecules demonstrate anchors at P3 or P5(Rammensee et al. 1997). The P2 anchor is assumed to associate with theB-pocket of the binding groove, while P9, the C-terminal residueassignment, associates with the F-pocket. “Auxiliary” or “secondary”anchors (alternative positions frequently defined by the occupancy ofchemically similar residues) are additionally included in the motifs ofsome class I molecules (Rammensee et al. 1997). In general, a commoninterpretation of this type of data is that endogenous peptide bindingand/or loading requires a nonamer with particular P2 and P9 anchors. Aswill be discussed, many searches (and consequent failed attempts) forputative viral or tumor class I-presented epitopes have subsequentlybeen predicated upon nonameric templates with P2 and P9 anchorassignments.

Broad efforts have been focused upon establishing and analyzing motifsfrom natural class I ligands (for example, Huczko et al. 1993;Fleischhauer et al. 1994; Kubo et al. 1994; Barber et al. 1995; Steinleet al. 1995; Barber et al. 1996; Tzeng et al. 1996; Tieng et al. 1997;Yagüe et al. 1998). However, motifs fail to reflect the true complexityof peptides presented by divergent class I molecules. Drawbacks areevident in that numerous characterized peptide sequences that bind classI are greater than 9 residues long, with 14 being the largest identifiedto date (Engelhard 1994); The binding of longer ligands likely resultsfrom stable associations at anchor positions coupled with centralprotrusion of the peptide outward from the groove (Fremont et al. 1992;Guo et al. 1992; Madden et al. 1993). Furthermore, other examinations ofspecific peptides naturally presented by class I MHC of both humans andmice have indicated that some fail to comply with their respectivelydefined motif anchors (Calin-Laurens et al. 1993; Henderson et al. 1993;Sadovnikova et al. 1993; Kawakami et al. 1994; Urban et al. 1994;Malarkannan et al. 1996; Mata et al. 1998), thus suggesting that bothlength variance and “nonanchor” residues in the peptide could playsignificantly more prominent roles in binding than strictly accountedfor by a given pooled motif alone. Studies have also indicated that lowcopy-number peptides, presented by only a small proportion of the totalclass I molecules expressed upon the cell surface, can successfullyelicit CTL responses (Cox et al. 1994; Malarkannan et al. 1996; Wang etal. 1997). Issues such as these clearly reflect the limitations posed inapplying Edman sequencing to the complex mixtures of peptides extractedfrom class I molecules (Stevanovic and Jung 1993). As a result, pooledEdman sequencing is therefore unable either to precisely characterizeindividual ligands or to effectively identify overlaps in ligandpresentation.

Shortly after the debut of examining class I ligands by pooled Edmansequencing, the first reports of ligand analyses via mass spectrometricsequencing of organic ions from similarly-prepared class I ligandextracts began to emerge (Henderson et al. 1992; Hunt et al. 1992;Huczko et al. 1993; Appella et al. 1995). The utilization of MS/MS on atriple quadrupole mass spectrometer provided for the precisecharacterization of individual constituents at sub-picomolar levels frompools of class I peptides, in contrast to the picomolar detection limitsof Edman analysis. Furthermore, LC/MS prior to this step allowed forcomplexity estimates to be made. For example, based upon thequantitation of 200 different peptides present within HLA-A*0201extracts, extrapolation with regard to the peptides detected versustheir respective contributions to the mass spectrometric TIC obtainedindicated that at least 1,000 and perhaps as many as 10,000 uniquepeptides are bound by this class I allotype (Hunt et al. 1992). Theability to characterize ligands as such additionally assisted in beingable to identify and sequence single specific epitopes from RP-HPLCfractions demonstrating biological activity via CTL assays (Rötzschke etal. 1990; Henderson et al. 1993; Kawakami et al. 1994; Skipper et al.1996; Simmons et al. 1997; Wang et al. 1997; Hogan et al. 1998; Paradelaet al. 1998). Examination of ligands by mass spectrometry was thereforean effective development in both starting to fill the gaps often presentin pooled motifs and expediting the classification of ligandspotentially bearing immunological significance.

However, the routine application of mass spectrometric techniques toclass I ligand examination has remained relatively isolated; it ispracticed in only a handful of laboratories (for example, Woods et al.1995; Simmons et al. 1997; Flad et al. 1998; van der Heeft et al. 1998;Yagüe et al. 1998). This appears largely due to the inherentdifficulties imposed in handling the small quantities of peptidesextracted for study (Henderson et al. 1993; Appella et al. 1995; van derHeeft et al. 1998) as well as the notably tedious nature of thesubsequent data processing (Papayannopoulos 1995; van der Heeft et al.1998). Other issues concern the specific instrumentation and its mode ofoperation, since: (i) the ability to consistently identify peptides fromparticular extracts relies upon dependable RP-HPLC gradients forseparation; and (ii) the possible ionization and/or detection interfacesfunction in distinct manners that can influence the data ultimatelyacquired (Chapman 1996; Watson 1997). In summary, understanding theimpact of polymorphism upon the binding of endogenous peptides hashistorically been limited by small amounts of ligands available foranalyses.

What has not been accomplished yet by mass spectrometry is thesystematic definition of peptides across diverse class I allotypes.Various in vitro assays have been developed to complement massspectrometric approaches. The assays are performed using a number ofdifferent protocols with the common theme of assessing the relativeabilities in vitro of synthetically defined peptides to associate withspecific class I α-chains and β₂m. In the case of binding assays,synthetic peptides as well as a peptide standard are incubated withpurified class I complexes and tested in their capacities tocompetitively displace the ligands already bound (Chen and Parham 1989;Ruppert et al. 1993; Sette et al. 1994). This has likewise beenperformed by stripping ligands from class I complexes expressed on thecell surface by acid treatment and then incubating the cells with thesynthetic peptides (Drijfhout et al. 1995). Another common approach tocompetitive binding assays involves FACS analysis with class I-specificMAbs after incubation of synthetic peptides with various cell linesincluding RMA-S (Townsend et al. 1989), T2 (Salter et al. 1985),Hmy2.C1R (Storkus et al. 1989), or 721.221 (Kavathas et al. 1980)transfected to express HLA α-chains (Huczko et al. 1993; Takamiya et al.1994; Zeh III et al. 1994; Boisgerault et al. 1996; Konya et al. 1997).Alternatively, de novo reconstitution assays can be performed byincubating synthetic peptides with free α-chains and β₂m and comparingthe resultant quantities of free versus complexed α-chains (Tanigaki1992; Fruci et al. 1993). Other methods of reconstitution have also beenapplied (Silver et al. 1991; Parker et al. 1994; Gnjatic et al. 1995;Robbins et al. 1995; Tan et al. 1997).

While these in vitro assays with synthetic ligands have led to thedevelopment of extensive “supermotifs”, or enhanced motifs that ascribebinding preferences to particular class I allotypes or groups of relatedallotypes (del Guercio et al. 1995; Sidney et al. 1996a; Sidney et al.1996b), they present obvious limitations. A technical concern is thatbinding/reconstitution determinations are often based upon either“standard” probe peptides for each allotype examined or meanexperimental values that can significantly differ from one laboratory orexperiment to another. A specific illustration is based on the resultsgenerated by three different groups testing HLA-A*0201 with panels ofsynthetic peptides; the assignment of residues preferred or deleteriousfor binding at given positions varied widely (Ruppert et al. 1993;Parker et al. 1994; Drijfhout et al. 1995). In fact, only three residuesimilarities carried across all three studies.

Ruppert and colleagues and Drijfout and colleagues each employedcompetitive binding assays; however, the former involved assessing theability of synthetic peptides to inhibit binding of a radioiodinatedstandard peptide to membrane-extracted class I complexes, while thelatter involved stripping peptides from class I complexes on B-LCLs anddetermining by FACS analysis the ability of synthetic peptides toinhibit binding of a different fluorescence-labeled standard. On theother hand, Parker and colleagues employed a reconstitution assay whichmeasured the incorporation of radioiodinated β₂m into complexes refoldedusing synthetic peptides and α-chains prepared from Escherichia coliinclusion bodies. Of perhaps greater significance, these types of assaysfail to account for the processing/loading physiology of trimolecularcomplex formation within the cell (Hogan et al. 1988); indeed,differences have been documented in performing comparative examinationsby pooled Edman sequencing and mass spectrometry uponnaturally-extracted versus artificially bound peptides (Davenport et al.1997). In one specific case, an immunodominant HIV peptide divulgedthrough other mechanisms completely failed to demonstrate binding to itsrestricting allotype via an in vitro assay (Tsomides et al. 1991).Conclusions regarding ligand presentation that are drawn frombinding/reconstitution assays thus cannot be applied generally withoutcaveat.

Thus, the present invention(s) aim to provide a methodology for theproduction of soluble MHC Class I and II molecules from either gDNA orcDNA starting material such that the structural and functional impact ofHLA class I polymorphism on peptide binding can be assessed and, inparticular, to test how natural ligand presentation overlaps exist invarying degrees across the polymorphisms of divergent class I bindinggrooves. Furthermore, the soluble MHC molecules can be used in thefunctional testing of CTL binding assays and vaccine development.Utilizing this specification, one of ordinary skill in the art will ableto: (i) generate ligands and hence ligand maps from the peptide poolsextracted from series of distinct yet related class I HLA-B15 allotypes;(ii) compare the different ligand maps to identify potentially sharedelements; and (iii) characterize the elements identified to positivelyor negatively validate the occurrence of overlapping ligands. One ofordinary skill in the art, given the present specification, will alsorealize that the ability to produce soluble MHC molecules from eithergDNA or cDNA starting material also will allow for other useful assayand vaccine development such as the functional testing of peptideligands of interest to determine if, when, and how such peptide ligandsprovoke and/or stimulate an immune response. All of which is directedtoward the goal of identifying candidate peptide ligands that may beused singly or together as a vaccine and/or immune system primer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a graphical representation of MHC class I location and heavychain coding region. (A) Simple map of the human MHC region,specifically highlighting the B, C, and A loci that encode the class Iheavy chains as simplified from (Janeway and Travers 1994). Geneticdistances are estimated in kilobases. Other genes (not shown) includingheavy chains and transporter/chaperone proteins (class II) andcomplement proteins and cytokines (class III) are encoded within theremaining MHC regions. (B) The basic exon structure of MHC class Itranscripts. A total of seven exons encode the leader peptide (1), α₁domain (2), α₂ domain (3), α₃ domain (4), transmembrane domain (5), andcytoplasmic domains (6-7). Specific exon regions are indicated inparentheses for HLA-B*15011 (accession number U03859, Hildebrand et al.1994), which encodes the HLA-B*1501 heavy chain.

FIG. 2 is a three-dimensional graphical representation of theconfiguration of the MHC class I extracellular domains. (A) The completeextracellular portion of the class I molecule HLA-B*3501, revealing thethree domains (α₁, α₂, and α₃) of the heavy chain as well as thenoncovalently associated light chain, β₂m. Top (B) and side (C) views ofthe basket-like antigen binding groove formed by α₁ and α₂; the α-helixand β-sheets for each domain are respectively indicated in (B). Thesethree images were rendered from the HLA-B*3501 Protein Data Bank(Brookhaven National Laboratory) crystal structure coordinate file, 1A1N(Smith et al. 1996a), using the Swiss-Pdb Viewer 3.01 software (Guex andPeitsch 1996). The CHO linked to α₁ residue 86 is not shown.

FIG. 3 is a graphical representation of the specificity determiningpockets of the MHC class I ligand binding groove. The structuralresidues contributing to the six pockets (A through F) of the antigenbinding groove formed by α₁ and α₂ are indicated in black and numberedfrom the N terminus of the mature class I α-chain. The residues are ascollectively defined in the literature (Saper et al. 1991; Matsumura etal. 1992; Chelvanayagam 1996). In terms of a nonameric ligand, thepockets are assumed to accommodate the amino acids occurring at givenligand positions as follows: A-pocket, P1; B-pocket, P2; C-pocket, P6;D-pocket, P3; E-pocket, P7; and F-pocket, P9. The majority of residuesthat contribute to these binding pockets are oriented such that they aresolvent-inaccessible in mature trimers.

FIG. 4 is a pictorial representation summarizing MHC class I trimerassembly and transport. The typical endogenous processing pathway shownis simplified from the description disclosed herein. Major proteinsparticipating in the processes are specifically illustrated andlabelled. Some alternative processing and/or transport mechanismscurrently under investigation are designated in the diagram by questionmarks. Assembled trimers travel through the Golgi complex prior tovesicular delivery to the surface membrane of the cell (not shown).

FIG. 5 is a schematic representation of the deduced contact regionbetween the MHC class I α₁/α₂ and TCR V_(α)/V_(β) interfaces. From thediagonal orientation of the estimated binding footprints of CDRs 1, 2,and 3 (numbered) for each of TCR-V_(α) and V_(β) with respect to the topof MHC-α₁ and α₂, the general interaction between TCR and MHC appears topredominantly involve V_(α)/V_(β) contact with both the class Isolvent-accessible helical residues and the central portions of thebound ligand (not shown).

FIG. 6 is a flow diagram of the overall strategy for comparativelymapping and characterizing peptide ligands presented by class I HLA. Theapproach taken to address the presence of overlapping ligands acrossdiverse class I molecules consisted of three basic parts: (A)sHLA-producing transfectant establishment and culture; (B) extraction,purification, and separation of ligands; and (C) ion mapgeneration/comparison and characterization of individual peptides.Though only two molecules are indicated for simplification, numerousadditional molecules (as indicated in the specification) weresimultaneously carried through the steps shown.

FIG. 7. Hypothesized evolutionary relationships of HLA-B15 allotypesaccording to expressed α-chain polymorphisms. Shown in this scheme arethe 46 allotypes whose amino acid sequences are provided in herein.Solid lines indicate single mutagenic events separating given allotypes.Dashed lines indicate that greater than one mutagenic event separatesgiven allotypes; intermediaries are indicated by question marks unlessmore specifically suspected (as in the case of a probable B*1539/B17recombinant). None of the line lengths are reflective of mutationalrates.

FIG. 8 is a graphical representation of the localization of antigenbinding groove substitutions distinguishing the B*1512, B*1508, B*1501,B*1503, B*1518, and B*1510 allotypes. The structural residues of theantigen binding groove formed by α₁ and α₂ which differ among the sixalleles according to Table 1 are indicated on the ribbon diagram inblack and numbered from the N terminus of the mature class I α-chain.

FIG. 9 is a graphical representation summarizing of the UnisynTechnologies CP-3000 basic flow path. The system was assembled andoperated as described herein. Arrows indicate unidirectional media flow.

FIG. 10 is two graphs showing sHLA production during bioreactor runswith two different B*1501 transfectants. Constructs using PSR_(α-neo)and pcDNA3 for producing soluble B*1501 were separately transfected intoB-LCL 721.221; subclones were then cultured in CP-3000 systems asdescribed herein. Harvests samples drawn on the days indicated along thex-axis during each run were subjected to ELISA, yielding therepresentative sHLA production data shown.

FIG. 11 is a graph showing RP-HPLC fractionation of peptides extractedfrom B*1510. The UV trace obtained during separation as described hereinof approximately 400 μg of peptides extracted from B*1510 reveals thebulk of absorbance to occur along the gradient (black line) between10-40% buffer B (indicated at the right). The three peaks associatedwith the control dye (methyl violet base B), as well as the primaryregion subjected to intensive mapping between B*1501, B*1503, B*1508,and B*1510 peptides (fractions 6-19), are indicated.

FIG. 12 is a schematic showing the generalized components of a triplequadrupole mass spectrometer. The basic constituents of the systemdescribed herein include: (A) an electrospray source/ionizationinterface for sample introduction; (B) three quadrupoles for ionmanipulation, which includes mass filtration (Q1 or Q3), transmission(all quadrupoles), and/or collision (q2); and (C) a detector foramplifying transmitted ion signals so that they can be recorded andanalyzed.

FIG. 13 includes two graphs (A and B) and chart C that show theidentification and verification of a ligand overlap across divergentHLA-B15 molecules. NanoES-MS spectral ion maps obtained individuallyfrom RP-HPLC fraction 8 for each of B*1501, B*1503, B*1508, and B*1510were aligned for comparison; an expanded view of the range 495-555 m/z,or amu, is shown in (A). The ion mass centered at 517.2 m/z matchedacross the spectra of B*1501, B*1503, and B*1508 (top three panels) butnot B*1510 (bottom panel). This ion was subsequently selected forNanoES-MS/MS from fraction 8 of the first three class I MHC molecules.The homologous spectra resulting from fragmentation of this [M+2H]²⁺ ion(B) classified the peptide as a positive match, or ligand overlap,occurring across B*1501, B*1503, and B*1508 and allowed for primarysequence derivation (C). N- and C-terminal peptide fragments present inall three NanoES-MS/MS spectra are labeled according to standardnomenclature (Roepstorff and Fohlman 1984) in the top panel of (B) andunderlined in (C); immonium ions are indicated by their single-letteramino acid codes in (B), and the sequences of internal cleavage productsare also specified.

FIG. 14 is a graphical representation showing the reproducibility of theclass I HLA ligand mapping and characterization strategy disclosedherein. Approximately 400 μg each of B*1508 ligands obtained from twoseparate bioreactor runs performed six months apart were fractionated byRP-HPLC as shown for B*1510 (FIG. 11). NanoES-MS ion mapping andcomparison were then performed as described herein. As illustrated bythe spectra of fraction 15 for each (A and B), the ion maps wereconsistent with one another; subjecting an ion (514.0) from each B*1508fraction to NanoES-MS/MS further demonstrated reliability of theprotocols employed.

FIG. 15 is a tabulation of the pooled Edman sequencing motifs forpeptides extracted from B*1501 purified by two different MAbs. Dataderived from the eluates of B*1501 complexes purified using either W6/32(A) or BBM.1 (B) indicated amino acid residues that had been previouslyattributed to the corresponding positions in B*1501 motifs (solidcharacters), as well as a residue which was not included in any previousmotif descriptions (Falk et al. 1995; Barber et al. 1996). Edmandegradation was carried out as described hereinafter, and relativepicomolar fold increases are indicated in parentheses to the right ofeach residue; amino acids demonstrating a 2.0 to 3.5-fold picomolarincrease over the previous degradation cycle were grouped as “strong”,while those demonstrating an increase of greater than 3.5-fold weregrouped as “dominant”. Dashes indicate that no distinct residues couldbe detected as either dominant or strong at the given positions.

FIG. 16 is a tabulation and corresponding pictorial representation ofpooled Edman sequencing motifs for peptides extracted from B*1508,B*1512, B*1503, B*1518, and B*1510. Edman degradation was carried out asdescribed herein, and the data were analyzed as described hereinbeforewith respect to FIG. 15. Ribbon diagrams of the class I antigen bindinggroove to the right of each motif show the structural residues of theα₁/α₂ antigen binding groove among each of the alleles which differ withrespect to B*1501 according to Table 1; they are indicated in black andnumbered from the N terminus of the mature class I α-chain.

FIG. 17 is a graphical showing RP-HPLC separation of ligands fromBBM.1-purified B*1501. The UV trace obtained during separation asdescribed herein of approximately 150 μg of peptides extracted fromBBM.1-purified B*1501 reveals the bulk of absorbance to occur along thegradient (black line) between 10-30% buffer B (indicated at the right).The fractions collected are numbered; although all fractions wereexamined, only those subsequently selected for analysis and datapresentation in FIGS. 18 and 19 are marked by dots. It is noted that thechosen fractions were distributed evenly across the entire region ofinterest and included a wide variety that were of high as well as low UVabsorbance and/or resolution.

FIG. 18 is four graphs showing the percentages of RP-HPLC ligandfractions from BBM.1-purified B*1501 demonstrating particular amino acidoccupancies by Edman degradation. Sequence complexity among the peptideswas summarized by averaging the frequency of occurrence for amino acidresidues at P2 through up to P12 for the fractions marked by dots inFIG. 17. Amino acids are grouped along the x-axis according to theirphysicochemical natures (charged, polar, or hydrophobic), asspecifically designated in the chart for P10 and indicated within allfour charts by differential shading. Residues observed at P2 and P9 informer B*1501 motif descriptions are indicated in bold italics. Since itwas not derivatized, cysteine was undetectable and is thereforeexcluded.

FIG. 19 is four tabular examples of Edman sequence complexity amongRP-HPLC ligand fractions from BBM.1-purified B*1501. While each of thefractions marked by dots in FIG. 15 was subjected to Edman sequencing,the results obtained from fractions 10, 15, 28, and 31 are shown here tospecifically illustrate some of the diverse trends observed amongconstituents of the ligand mixture. Dominant and strong assignments weremade as described hereinbefore for FIG. 13; weak assignments were madefor amino acids demonstrating a 1.5 to less than 2.0-fold picomolarincrease over previous cycles. No assignments are shown for P1 due tolower confidence since comparison with former cycles was impossible. Thepositional assignments in fraction 15, which are identified in bolditalics, appear to correspond to a hexamer, IAVGYV, derived from HLAclass I α-chain₂₃₋₂₈ (Prilliman et al. 1998).

FIG. 20 is five graphs summarizing the length diversity among the 449characterized HLA-B15 ligands. Graphed data from the ligands listed inTables A-E summarizes length diversity among the peptides respectivelycharacterized from B*1501, B*1503, B*1508, B*1510, and B*1512.

FIG. 21 is two graphs showing N- and C-regional diversity observedthrough alignments of B*1501 ligands. Frequency of occurrence for aminoacids at the first (A) and final (B) four positions among the ligandscharacterized from B*1501 (Table A). The graphs were generated fromseparate N- and C-terminal data alignments.

FIG. 22 is two graphs showing N- and C-regional diversity observedthrough alignments of B*1503 ligands. Frequency of occurrence for aminoacids at the first (A) and final (B) four positions among the ligandscharacterized from B*1503 (Table B). The graphs were generated fromseparate N- and C-terminal data alignments.

FIG. 23 is two graphs showing N- and C-regional diversity observedthrough alignments of B*1508 ligands. Frequency of occurrence for aminoacids at the first (A) and final (B) four positions among the ligandscharacterized from B*1508 (Table C). The graphs were generated fromseparate N- and C-terminal data alignments.

FIG. 24 is two graphs showing N- and C-regional diversity observedthrough alignments of B*1510 ligands. Frequency of occurrence for aminoacids at the first (A) and final (B) four positions among the ligandscharacterized from B*1510 (Table D). The graphs were generated fromseparate N- and C-terminal data alignments.

FIG. 25 is two graphs showing N- and C-regional diversity observedthrough alignments of B*1512 ligands. Frequency of occurrence for aminoacids at the first (A) and final (B) four positions among the ligandscharacterized from B*1512 (Table E). The graphs were generated fromseparate N- and C-terminal data alignments. The information shown herefor B*1512 ligands is skewed in relation to that obtained for ligandsfrom the other four allotypes since (i) a comparatively fewer B*1512peptides were sequenced, and (ii) over half of the B*1512 peptidescharacterized were specifically investigated as ion map differences withB*1501.

FIG. 26 is a graphical and tabular representation showing ligandoverlaps identified by NanoES-MS mapping and characterized byNanoES-MS/MS during comparative analysis of B*1508, B*1501, B*1503, andB*1510 extracts. Ribbon diagrams of the class I antigen binding grooveindicate residue substitutions (black, numbered from the first residueof the mature α-chain) between each B15 molecule with respect to B*1501.The peptides are categorized into three different groups from top tobottom as follows: ligands that overlap B*1508 and B*1501; ligands thatoverlap B*1508, B*1501, and B*1503; and ligands that overlap B*1501 andB*1503. Dashes represent positions at which amino acids could not beunambiguously assigned through the NanoES-MS/MS fragmentation patternsand/or Edman data obtained. Ligand residues that coincide with dominantand strong amino acids for the given motifs (FIGS. 15 and 16) areindicated in bold.

FIG. 27 is a pictorial representation of the proposed N-proximal andC-terminal anchoring of a nonamer overlapping B*1508, B*1501, andB*1503. The shared C-terminal anchoring preferences for Tyr in theNQZHGSAEY ligand among B*1508, B*1501, and B*1503 as defined by theirrespective motifs (FIGS. 15 and 16) are shaded black, while the variedN-proximal anchoring preferences likewise reflected in the motifs arecross-hatched. Ligand residues are numbered sequentially from the Nterminus.

FIG. 28 is a pictorial schematic summarization the summary ofstructure-function relationships among the HLA-B15 allotypes from whichligands were characterized. HLA-B15 allotypes for which a motif is knownare indicated in bold, while allotypes with currently undefined motifsare indicated in italics. Arrows are representative of single mutagenicevents (which are specifically defined for each arrow); bold arrowsemphasize evolutionary pathways between structures for which motifs havebeen described either here or elsewhere. P2 and P9 designations arelisted. Tentative designations have been made for the italicizedallotypes by extrapolation from known motifs in light of thepolymorphisms present. P2/P9 specificities for B*1519 are based uponB*1512. P2/P9 specificities for B*1539 are based upon what has beenobserved between B*4402 and B*4403 (Fleischhauer et al. 1994). P2/P9specificities for B*1529 are based upon B*1508. P2/P9 specificities forB*1523 are based upon what has been observed between B*1502 and B*1513(Table 7), as well as observations between B*0801 and B*0802 (Arnett etal. 1998). P2/P9 specificities for B*1547 are based upon therelationship previously discussed between B*1503 and B*4801(Martinez-Naves et al. 1997). P2/P9 specificities for B*1537 are basedupon B*1402 (DiBrino et al. 1994). The three sections (A, B, and C) intowhich the molecules have been divided are discussed in the text. Forreference, a ribbon diagram of the class I antigen binding grooveindicating residue substitutions (black, numbered from the first residueof the mature α-chain) between the molecules with respect to B*1501 isprovided.

FIG. 29 is a pictorial representation of the PCR strategy for theproduction of soluble HLA from gDNA according to the methods of thepresent invention.

FIG. 30 is a gel image of the primary PCR of 3A394.

FIG. 31 is a gel image of the secondary PCR of 3A394.

FIG. 32 is a gel image showing 3A394 and pcDNA3.1 digested with EcoR Iand Xba I.

FIG. 33 is a gel image showing the restriction digests of 3A394 clones.

FIG. 34 is a graph showing the comparative binding of three monoclonalantibodies to four soluble HLA molecules.

FIG. 35 is a pictorial representation of the MHC.

FIG. 36 is a pictorial representation of an HLA molecule.

FIG. 37 is a pictorial representation of the HLA binding groove thatbinds antigenic peptides.

FIG. 38 is a pictorial representation of the antigen processing andassembly of the MHC class I/peptide complex.

FIG. 39 is a pictorial representation of HLA peptide loading andmovement of the HLA molecule to the cell surface.

FIG. 40 is a pictorial representation of a tetramer biotinylated sHLAcomplex of the present invention.

FIG. 41 is a pictorial representation of a bacterial expression vectorencoding a biotinylation substrate peptide sequence.

FIG. 42 is a pictorial representation of the biotinylation of sHLA.

FIG. 43 is a pictorial representation of the conjugate used to confirmbiotinylation of the sHLA molecule.

FIG. 44 is a graph showing production of sHLA-B*0702 by T₂ transfectantsafter peptide pulsing.

FIG. 45 is a graph showing the elution curve of the Elisa assay used toconfirm sHLA production.

FIG. 46 is a graph showing that an increase in time results in greaterbiotinylation.

FIG. 47 is a graph showing separation of biotinylated class I from freebiotin.

Table 1. Amino acid differences among the B*1512, B*1508, B*1501,B*1503, B*1518, and B*1510 allotypes. Residues, noted across the top ofthe table, are numbered from the N terminus of the mature class I heavychain for each; the 24 amino acid leader sequence is therefore notincluded. The residue positions are differentially highlighted in black(α₁) or gray (α₂). Positions of identity with the consensus sequence(italicized) drawn from B*1501 are indicated by dashes.

Table 2. PCR and sequencing primers for creating and verifying sHLAconstructs. Designated restriction enzyme cut sites are underlined onthe 5′ and 3′ PCR primers, and the regions of the 3′ PCR primers thatinserted a stop codon at position 300 are shown in bold italics.Sequencing primers were Cy5-labeled; the regions that they eithersequenced through or hybridized with are indicated in parentheses.

Table 3. Additional P2, P9, and length variability revealed throughEdman sequencing of RP-HPLC ligand fractions from W6/32-purified B*1501,B*1508, B*1503, and B*1510. The number of fractions for each which weresubjected to Edman degradation as described herein and shown in FIG. 13are shown; ligands present in the fractions from all four moleculesdemonstrated evidence of (i) expanded P2 and P9 occupancy, and (ii)sequence beyond the 9 residues described by their respective motifs (asshown in FIGS. 15 and 16).

Table 4. HLA-B15 ligands identically matching database source proteins,by category. The ligands are categorized according to source proteinfunctions. Residues for the specific ligands are numbered from theinitiating Met of their respective source proteins. Ligands from HLA-B15molecules not studied here (B*1502, B*1509, and B*4601) are referencedherein.

Table 5. Ligands unique to B*1512, as compared with B*1501.Positively-charged P1 residues are indicated in bold. Dashes representpositions at which amino acids could not be unambiguously assignedthrough the NanoES-MS/MS fragmentation patterns and/or Edman dataobtained. Underlined residues designate tentative assignments.

Table 6. Ligand binding groove residues interacting with the B-pocketamong HLA-B15 allotypes bearing known Edman-derived motifs. Residues,noted across the top of the table, are numbered from the N terminus ofthe mature class I heavy chain for each. The residue positions aredifferentially highlighted in black (α₁) or gray (α₂); vertical hatchingimmediately below further distinguishes those residues located on ahelix. The molecules are grouped according to dominant/strong P2 motifsimilarities. Structural residues specifically discussed in the text areindicated in bold italics.

Table 7. Ligand binding groove residues interacting with the F-pocketamong HLA-B15 allotypes bearing known Edman-derived motifs. Residues,noted across the top of the table, are numbered from the N terminus ofthe mature class I heavy chain for each. The residue positions aredifferentially highlighted in black (α₁) or gray (α₂); vertical hatchingimmediately below further distinguishes those residues located on ahelix. The molecules are grouped according to dominant/strong P9 motifsimilarities. Structural residues specifically discussed herein areindicated in bold italics.

Table 8. B*1510 ligands exhibiting Pro with Ala and/or Val in variousC-proximal combinations. C-proximal occurrences of Pro, Ala, and Val areindicated in bold. Dashes represent positions at which amino acids couldnot be unambiguously assigned through the NanoES-MS/MS fragmentationpatterns and/or Edman data obtained. Underlined residues designatetentative assignments.

Table 9. B*1501 ligands: N- and C-regional occupancies observed at >10%among 126 ligands. The positional occupancy percentages for residuesamong the ligands from Table A greater than 10% are specifically shown;dashes indicate occupancy rates below 10% for the given side chains.N-regional positions and N_(sum) values are highlighted in black, whileC-regional positions and C_(sum) values are highlighted in gray.

Table 10. B*1503 ligands: N- and C-regional occupancies observed at >10%among 74 ligands. The positional occupancy percentages for residuesamong the ligands from Table B greater than 10% are specifically shown;dashes indicate occupancy rates below 10% for the given side chains.N-regional positions and N_(sum) values are highlighted in black, whileC-regional positions and C_(sum) values are highlighted in gray.

Table 11. B*1508 ligands: N- and C-regional occupancies observed at >10%among 96 ligands. The positional occupancy percentages for residuesamong the ligands from Table C greater than 10% are specifically shown;dashes indicate occupancy rates below 10% for the given side chains.N-regional positions and N_(sum) values are highlighted in black, whileC-regional positions and C_(sum) values are highlighted in gray.

Table 12. B*1510 ligands: N- and C-regional occupancies observed at >10%among 123 ligands. The positional occupancy percentages for residuesamong the ligands from Table D greater than 10% are specifically shown;dashes indicate occupancy rates below 10% for the given side chains.N-regional positions and N_(sum) values are highlighted in black, whileC-regional positions and C_(sum) values are highlighted in gray.

Table 13. B*1512 ligands: N- and C-regional occupancies observed at >10%among 30 ligands. The positional occupancy percentages for residuesamong the ligands from Table E greater than 10% are specifically shown;dashes indicate occupancy rates below 10% for the given side chains.N-regional positions and N_(sum) values are highlighted in black, whileC-regional positions and C_(sum) values are highlighted in gray.

Table 14. Overlaps identified through RP-HPLC/NanoES-MS ligand massmapping and characterized by NanoES-MS/MS. Dashes represent positions atwhich amino acids could not be unambiguously assigned through theNanoES-MS/MS fragmentation patterns and/or Edman data obtained.Underlined residues designate tentative assignments.

Table 15. Overlap frequencies observed between B*1501 and B*1512,B*1508, B*1503, or B*1510. The frequencies of ligand presentationoverlap for the molecules listed with respect to B*1501 were determinedfrom the number of total matching ion masses subjected to NanoES-MS/MSand the number of those ions collided analyzed by NanoES-MS/MS confirmedas actual overlaps. The resulting values are conservative since not allions selected for examination successfully yielded fragments that couldbe used for evaluating the sequence positivity of mass matches.

Table 16. Potential B*1501 epitopes selected from the EBV gp85structural antigen. The EBV gp85 protein (accession number 1334905,Arrand et al. 1981) was manually scanned for epitopes with considerationto B*1501. In the first column, epitope candidates matching the lengthand sequence constraints of the B*1501 pooled motif (FIG. 15) arelisted. In the second column, epitope candidates matching the B*1501motif-prescribed P2 and C-terminal occupancies but demonstrating relaxedlength constraints (7 to 11 residues, according to the B*1501 panel inFIG. 20) are listed. In the third column, epitope candidates matchingthe B*1501 motif-prescribed nonameric length but demonstrating P2flexibility (according to FIG. 21A) are listed.

Table 17 PCR of sHLA from gDNA primers.

Table 18 clone sequencing 5′CYS primers.

Table 19 optical density readings and concentration of DNA extractedfrom sample 3A394.

Table 20 optical density readings of positive clones.

Table 21 optical density readings of AF/102 plasmid extracted.

Table 22 viability of cells after two (2) days.

Table 23 Elisa results for positive transfectants exhibiting G418resistance.

Table 24 HLA types and sHLA molecules produced.

Table A derived peptide ligands from B*1501.

Table B derived peptide ligands from B*1503.

Table C derived peptide ligands from B*1508.

Table D derived peptide ligands from B*1510.

Table E derived peptide ligands from B*1512.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail byway of exemplary drawings, experimentation, results, and laboratoryprocedures, it is to be understood that the invention is not limited inits application to the details of construction and the arrangement ofthe components set forth in the following description or illustrated inthe drawings, experimentation and/or results. The invention is capableof other embodiments or of being practiced or carried out in variousways. Also, it is to be understood that the phraseology and terminologyemployed herein is for the purpose of description and should not beregarded as limiting.

As described hereinabove, characterizing naturally processed HLA class Iand class II ligands is a key element behind the basic understanding ofhow polymorphism impacts ligand presentation. However, technical andscientific challenges including both extreme sample heterogeneity andlimited sample sizes complicate such examinations. Thousands of distinctpeptides are present within a ligand extract prepared from a single typeof class I molecule, and the immunoprecipitation/extraction protocolstypically employed to recover peptide ligands yield sparse quantities onthe order of ˜20 μg (Hunt et al. 1992; Henderson et al. 1993). Thesefactors often require specialized biochemical expertise not necessarilyavailable in either the common laboratory or core facility.

Class I major histocompatibility complex (MHC) molecules, designated HLAclass I in humans, bind and display peptide antigens upon the cellsurface. The peptides they present are derived from either normalendogenous proteins (“self”) or foreign proteins (“nonself”), such asproducts of malignant transformation or intracellular pathogens such asviruses. In this manner, class I molecules convey information regardingthe internal fitness of a cell to immune effector cells including butnot limited to CD8⁺ cytotoxic T lymphocytes (CTLs), which are activatedupon interaction with “nonself” peptides and which lyse or kill the cellpresenting such “nonself” peptides.

Class II MHC molecules, designated HLA class II in humans, also bind anddisplay peptide antigens upon the cell surface. However, unlike class IMHC molecules which are expressed on virtually all nucleated cells,class II MHC molecules are normally confined to specialized cells, suchas B lymphocytes, macrophages, dendritic cells, and other antigenpresenting cells which take up foreign antigens from the extracellularfluid via an endocytic pathway. Therefore, the peptides they bind andpresent are derived from extracellular foreign antigens, such asproducts of bacteria that multiply outside of cells, wherein suchproducts include protein toxins secreted by the bacteria that havedeleterious and even lethal effects on the host. In this manner, classII molecules convey information regarding the fitness of theextracellular space in the vicinity of the cell displaying the class IImolecule to immune effector cells including but not limited to CD4⁺helper T cells, which help eliminate such pathogens both by helping Bcells make antibodies against microbes as well as toxins produced bysuch microbes and by activating macrophages to destroy ingestedmicrobes.

Class I and class II HLA molecules exhibit extensive polymorphism, whichis generated by systematic recombinatorial and point mutation events; assuch, hundreds of different HLA types exist throughout the world'spopulation, resulting in a large immunological diversity among thepopulation. Such extensive HLA diversity in the population results intissue or organ transplant rejection between individuals as well asdiffering susceptibilities and/or resistances to infectious diseases.HLA molecules also contribute significantly to autoimmunity and cancer.Because HLA molecules mediate most, if not all, adaptive immuneresponses, HLA proteins are needed to study transplantation,autoimmunity, and for developing vaccines.

There are several applications in which purified, individual class I andclass II MHC proteins would be highly useful. Such applications includeusing MHC-peptide multimers as immunodiagnostic reagents for diseaseresistance/autoimmunity; assessing the binding of potentiallytherapeutic peptides; elution of peptides from MHC molecules to identifyvaccine candidates; screening transplant patients for preformed MHCspecific antibodies; and removal of anti-HLA antibodies from a patient.Since every individual has different MHC molecules, the testing ofnumerous individual MHC molecules is a prerequisite for understandingdifferences in disease susceptibility between individuals. Therefore,purified MHC molecules representative of the hundreds of different HLAtypes existing throughout the world's population are highly desirablefor unraveling disease susceptibilities and resistances and fordesigning therapeutics.

Currently there is no readily available source of individual HLAmolecules. Until now, the quantities of HLA protein available were smalland typically consist of a mixture of different HLA molecules.Production of HLA molecules traditionally involves growth and lysis ofcells expressing multiple HLA molecules. Ninety percent of thepopulation is heterozygous at each of the HLA loci; codominantexpression results in multiple HLA proteins expressed at each HLA locus.To purify native class I or class II molecules from mammalian cellsrequires time-consuming and cumbersome purification methods, and sinceeach cell typically expresses multiple surface-bound HLA class I orclass II molecules, HLA purification results in a mixture of manydifferent HLA class I or class II molecules. When performing experimentsusing such a mixture of HLA molecules or performing experiments using acell having multiple surface-bound HLA molecules, interpretation ofresults cannot directly distinguish between the different HLA molecules,and one cannot be certain that any particular HLA molecule isresponsible for a given result. Therefore, a need exists in the art fora method of producing substantial quantities of individual HLA class Ior class II molecules so that they can be readily purified and isolatedindependent of other HLA class I or class II molecules. Such individualHLA molecules, when provided in sufficient quantity and purity, wouldprovide a powerful tool for studying and measuring immune responses.

The present invention envisions a method of producing MHC moleculeswhich are secreted from mammalian cells in a bioreactor unit.Substantial quantities of individual MHC molecules are obtained bymodifying class I or class II molecules so they are secreted. Secretionof soluble MHC molecules overcomes the disadvantages and defects of theprior art in relation to the quantity and purity of MHC moleculesproduced. Problems of quantity are overcome because the cells producingthe MHC do not need to be detergent lysed or killed in order to obtainthe MHC molecule. In this way the cells producing secreted MHC remainalive and therefore continue to produce MHC. Problems of purity areovercome because the only MHC molecule secreted from the cell is the onethat has specifically been constructed to be secreted. Thus,transfection of vectors encoding such secreted MHC molecules into cellswhich may express endogenous, surface bound MHC provides a method ofobtaining a highly concentrated form of the transfected MHC molecule asit is secreted from the cells. Greater purity can be assured bytransfecting the secreted MHC molecule into MHC deficient cell lines.

Production of the MHC molecules in a hollow fiber bioreactor unit allowscells to be cultured at a density substantially greater thanconventional liquid phase tissue culture permits. Dense culturing ofcells secreting MHC molecules further amplifies the ability tocontinuously harvest the transfected MHC molecules. Dense bioreactorcultures of MHC secreting cell lines allow for high concentrations ofindividual MHC proteins to be obtained. Highly concentrated individualMHC proteins provide an advantage in that most downstream proteinpurification strategies perform better as the concentration of theprotein to be purified increases. Thus, the culturing of MHC secretingcells in bioreactors allows for a continuous production of individualMHC proteins in a concentrated form.

Although class I and class II ligands were first examined by Edmansequencing, the primary characterization of individual ligands has beensignificantly improved upon through MS/MS applications. This isfrequently performed via LC/MS using a microcapillary (<1 mm i.d) HPLCcolumn directly interfaced with a triple quadrupole mass spectrometer(Hunt et al. 1992). The rationale behind using columns of small i.d. isthat a lower solvent flow rate is permitted that ultimately increasesthe sensitivity of mass spectrometric detection (Tomer et al. 1994).

Unfortunately, since sample load capacity decreases proportionately tothe column diameter, such columns and LC/MS methods can be technicallydifficult or inconsistent for laboratories not routinely employing themto operate, more robust protocols for producing and studying classI-derived peptides have been desired. The methodology of the presentinvention, as described herein, solves and/or meets this need in the artby a methodology which increases the quantities of ligands extractableby producing recombinant soluble class I and class II molecules. Thesample amounts subsequently available offset handling losses at thebench, which have been estimated at 50% (Veronese et al. 1996; Hogan etal. 1998; and Dr. Peter Parham, unpublished observations), and obviatethe need for microcapillary LC/MS prior to MS/MS analysis. This isbecause significantly larger (20-fold) ligand samples are insteadseparated by standard offline RP-HPLC followed by NanoES-MS mapping andNanoES-MS/MS sequencing. The methodology of the present invention hasproven consistent for comparatively examining peptides extracted fromdifferent class I molecules. As shown in FIG. 6, utilizing theproduction of soluble class I and class II molecules of the presentinvention, allows one of ordinary skill in the art to locate andcharacterize overlapping ligands among distinct allotypes (FIG. 6).

The initial HLA molecules selected for examination and production werefrom the HLA-B15 family, a hypothetical schematic of which is presentedin FIG. 7. The HLA-B15 family represents a broad and diverse group ofmolecules comprised of nearly 50 evolutionarily related allotypesdiffering almost sequentially by 1-15 peptide binding groove residues,and they are observed throughout numerous ethnic populations (Hildebrandet al. 1994); serological and DNA-based typing thus far confirmdistribution of B15 alleles among Caucasians, Amerindians (North andSouth), Mexicans, Blacks (African and American), Indians, Iranians,Pakistanis, Chinese, Japanese, Koreans, and Thais. The majority of HLAB-locus polymorphisms known to exist are represented among the membersof this allelic family. HLA-B*1501 appears to be the “ancestral allele.”

The specific B15 allotypes initially selected for review and use withthe present invention were B*1501 (Pohla et al. 1989; Choo et al. 1993;Hildebrand et al. 1994; Lin et al. 1996), B*1503 (Domena et al. 1993),B*1508 (Hildebrand et al. 1994), B*1510 (Domena et al. 1993; Rodriguezet al. 1993; Rodriguez et al. 1996), B*1512 (Hildebrand et al. 1994),and B*1518 (formerly B*7901, Choo et al. 1991; Lin et al. 1996;Rodriguez et al. 1996) (Table 1 and FIG. 8). B*1508 differs from B*1501by a single mutagenic event in the α₁ helix, while B*1512 differs by asingle mutagenic event in the α₂ helix; the remaining three allelesdemonstrate a progressive series of polymorphisms throughout theirbinding grooves imposed by sequential mutagenic events during theirdivergent evolution from B*1501.

Using the primer sets of HLA5UT (Domena et al. 1993) and sHLA3™(Prilliman et al. 1997) or 5PXI and 3PEI (Table 2) and template DNA fromreliable full-length cDNA clones of HLA-B15 molecules B*1501, B*1503,B*1508, B*1510, B*1512, and B*1518, truncating PCR was performed foreach on a Robocycler (Stratagene) for 30 cycles as previously described(Prilliman et al. 1997). The resultant PCR products contained the leaderpeptide, α₁, α₂, and α₃ coding domains of the HLA heavy chain.

The PCR products were introduced into mammalian expression vectors.Initial constructs (truncated B*1501, B*1503, and B*1508) were preparedwith the PSRα-neo vector (Lin et al. 1990), which has formerly been usedto express non-truncated HLA molecules (Barber et al. 1997;Martinez-Naves et al. 1997), while other constructs (truncated B*1501,B*1503, B*1508, B*1510, B*1512, and B*1518) were additionally preparedwith either pcDNA3 or pcDNA3.1 (−) (Invitrogen). Constructs using thePSRα-neo vector were made from PCR products of the HLA5UT and sHLA3™primers; the PCR products were subcloned into M13 (mp18 or mp19)according to standard protocols (Domena et al. 1993) so thatconfirmatory single-stranded DNA sequencing could be performed withCy5-labelled versions of the primers M13 universal, 4N, and 3N (mp 18)or M13 universal, 3S, and JD3S (mp 19), listed in Table 2 and describedpreviously (Ennis et al. 1990; Domena et al. 1993), using the AutoLoadsequencing kit and ALFexpress automated sequencer (both AmershamPharmacia Biotech). The insert was then prepared and purified, and thiswas followed by subcloning into PSRα-neo. Constructs using the pcDNA3vector were made from PCR products of the 5PXI and 3PEI primers; thesePCR products were subcloned into M13 and sequenced as above, followingwhich the insert was subcloned into pcDNA3. Constructs using thepcDNA3.1(−) vector were made from products of the 5PXI and 3PEI primers;PCR products were directly subcloned into pcDNA3.1 (−), following whichdouble-stranded DNA sequence analysis was performed with Cy5-labelledversions of the primers 3S, 4N, T7 promoter, and pcDNA3.1/BGH (Table 2).

DNA from each of the construct clones was prepared using Qiagen Midikits for transfection of the class I-negative B-LCL 721.221. Cellsgrowing in log phase in RPMI-1640+2 mM L-glutamine+phenol red+20% FCSwere pelleted and electroporation was performed as described (Gumperz etal. 1995) prior to beginning selection with 1.5 mg/mL G418. Uponestablishment of confluent growth after approximately 3 weeks, putativetransfectant wells were screened for sHLA production using a sandwichELISA (Prilliman et al. 1997). Transfectant wells positive for sHLAproduction were then subcloned by limiting dilution to establish celllines optimally secreting greater than 1 μg/mL of class I molecules instatic culture over 48 h. Satisfactorily subcloned transfectants werethen expanded, frozen in RPMI-1640+20% FCS+10% DMSO, and stored at −135°C.

Since hollow-fiber bioreactors have been applied in place of in vivohybridoma culture and MAb harvest from ascites (Evans et al. 1996) inorder to continuously produce large quantities of pure immunoglobulins,they were utilized to produce and harvest the sHLA of the presentinvention. The Unisyn Technologies CP-3000, the standard flow path andprimary components of which are diagrammatically simplified in FIG. 9,was selected for hollow-fiber bioreactor culture of successfullyestablished transfectants. In this system, basal media is pumped intothe fully-assembled system from a 200 L barrel; the media flows from the4 L reservoir tank into the hollow-fiber networks of the fourbioreactors, which provide 2.7 m² of surface area per cartridge, andthen exits as waste. ECS media and sHLA harvest are tandemly pumped intoand out of the 270 mL cartridges, respectively, with each bioreactorreceiving/yielding equal media/harvest volumes as regulated by in-linesolenoids.

The CP-3000 was set up according to the manufacturer's protocol. Afterthe system was completely prepared, at least 1×10⁹ viable cells of atransfectant were grown in roller bottles of RPMI-1640+2 mML-glutamine+phenol red+10% FCS. The cells were pelleted and inoculatedinto the ECS of the bioreactor cartridges. ECS feed and harvest bottleswere then attached to their corresponding lines, and the basal andrecirculation rates were initially set to 100 and 1000 mL/h,respectively; the ECS was usually not activated until 24-36 h followinginoculation. The system was then monitored at least twice daily over 4-6weeks, with adjustments made as necessary. This involved checking theglucose concentration and pH from manually-drawn reservoir samples,checking DO readings, and regulating the basal and ECS rates accordingly(Prilliman et al. 1997; Prilliman et al. 1998). A fresh harvest samplewas periodically extracted directly from the system to quantitate sHLAproduction by ELISA for production level monitoring (FIG. 10).

Cells were cultured in the bioreactor system during each run until thedesired amount of sHLA had been produced and collected in harvests. Eachof the bioreactor runs chronologically depicted in FIG. 10 was abortedonce approximately 150 mg of sHLA was determined by ELISA to becontained in the respective harvests collected (Prilliman et al. 1997).The majority of runs were performed using 721.221 cells transfected withpcDNA3 or pcDNA3.1 (−) vector constructs, considering: (i) thesignificant differences in time frame between the runs performed usingcell lines expressing soluble B*1501 from either the PSRα-neo or pcDNA3vectors (3 months versus 1 month); and (ii) the fact that peptidesextracted during each run produced identical motif results.

Upon completing a bioreactor run, sHLA complexes were purified from theharvests obtained (Prilliman et al. 1997). A 100 mL matrix of either theβ₂m-specific MAb BBM. 1 (Brodsky et al. 1979) or W6/32 (Barnstable etal. 1978) coupled to CNBr-activated Sepharose 4B (Amersham PharmaciaBiotech) according to the manufacturer's instructions was equilibratedwith wash buffer (20 mM sodium phosphate, pH 7.2+0.02% sodium azide),and harvests were applied to the column using a GradiFrac LC system(Amersham Pharmacia Biotech); the load capacities for 100 mL matrices ofthe MAbs BBM.1 and W6/32 were approximated at 10 and 40 mg sHLArespectively, as monitored for saturation ELISA of screening pre- andpost-column samples. The column was then washed, eluted with 0.2 Nacetic acid, and neutralized with wash buffer. Both BBM.1 and W6/32 wereused to affinity purify B*1501, the first molecule prepared. However,due to the differences in purification efficiency noted above betweenthe two MAbs, W6/32 alone was employed to isolate the remainingmolecules from bioreactor harvests. This MAb has been frequently used byothers in purifying HLA (Falk et al. 1991; Barber et al. 1997).

The fractions collected during affinity column elution demonstrating UVabsorbance at 280 nm were pooled, and glacial acetic acid was added to10% volume to extract the peptides as described (Barber et al. 1995).Bound peptides were separated from heavy chains, β₂m, and BSA by passagethrough 3 kDa exclusion membrane filters (Amicon) (Prilliman et al.1997). The ligand-containing eluate was then lyophilized.

To remove residual salts and free amino acids remaining from theextraction process, isolated peptides were purified of free amino acidsand salts prior to fractionation. This was done on a 2.1×100 mm C18column (Vydac) with a steep RP-HPLC gradient using a DYNAMAX HPLC system(Rainin). The gradient was generated by increasing to 100% buffer B(0.06% TFA in 100% acetonitrile) in 1 min, holding for 10 min, andreturning to buffer A (0.1% TFA in HPLC-grade water) in 1 min. Thecolumn was loaded with peptides reconstituted in the minimum volume ofbuffer A required for solubilization. During the run, the regioncorresponding to absorbance at 214 nm was manually collected. Typically1/100th of the total purified ligand collection volume was removed andsubjected to Edman sequencing for 14 cycles on a 492A pulsedliquid-phase protein sequencer (Perkin-Elmer Applied BiosystemsDivision) according to established protocols (Falk et al. 1991; Barberet al. 1995).

The purified ligands were next fractionated by RP-HPLC. For preliminarydiversity assessment, approximately 150 μg of peptides, as calculatedfrom the ELISA-based total mass of sHLA bound to the affinity column andan estimated 50% handling loss, were loaded in 10% acetic acid onto a1.0×150 mm C18 column (Michrom Bioresources, Inc.) and separated usingan initial gradient of 2-10% buffer B (0.085% TFA in 95% acetonitrile)in 0.02 min followed by a linear gradient of 10-60% buffer B in 60 minat 40 μL/min on a 2.1×150 mm C18 column (Michrom Bioresources, Inc.);buffer A was 0.1% TFA in 2% acetonitrile. Absorbance was monitored at214 nm, and fractions were automatically collected every minute. Forcomparative analyses, approximately 400 μg of peptides were injected in10% acetic acid containing 2 μg of the dye methyl violet base B tocontrol for gradient consistency between runs (FIG. 11). The gradientformation parameters consisted of 2-10% buffer B in 0.02 min and 10-60%buffer B in 60 min at 180 μL/min. Absorbance was monitored at 214 nm,and fractions were automatically collected every minute. Edmansequencing of fractions, when performed, was conducted on 1/20th ofeach.

To map extracted peptides and obtain primary sequences, a triplequadrupole mass spectrometer with an ES ion source, as genericallydepicted in FIG. 12, was employed. By using a triple quadrupoleinstrument, not only are all of the ions present within a given fractionbe summarized for a designated mass range (mass mapping), but ions maythen be selectively fragmented in order to obtain information from whichsequence information can be derived (characterization). This is due tothe flexibility afforded by the quadrupole mass analyzers: Q1 and Q3 actas mass filters which can be set to generate alternating DC and RFvoltage fields for selectively transmitting specific ions (Watson 1997).However, q2 is an enclosed transmission-only quadrupole; it can bepressurized with inert gas for the collisional dissociation of an iontransferred through Q1. The specific ionization interface, NanoES,chosen here as an ES source functions on the principles described bydevelopers Wilm and Mann (Wilm and Mann 1996). To establish and validatethe procedure, comprehensive peptide mapping and sequencing were firstperformed among fractions 6 through 19 (FIG. 11), which represented aregion of relatively rich ligand concentration (data not shown), forB*1501, B*1503, B*1508, and B*1510; once this was accomplished, a morefocused, and therefore less extensive, comparison was subsequently madebetween B*1501 and B*1512.

Prior to NanoES-MS, RP-HPLC fractions were completely dried by speedvac; the peptides were then resuspended in 0.1% acetic acid in 1:1methanol:water. Aliquots from each of the individually concentratedfractions were loaded into 5 cm gold/palladium alloy-coated borosilicatepulled glass NanoES sample capillaries (Protana A/S). To begin sampleflow and data collection, the loaded capillary tube was next carefullyopened as described (Wilm and Mann 1996). The capillary was thenpositioned directly in front of the API III⁺ (PE SCIEX) triplequadrupole mass spectrometer's orifice, and 20-30 scans were collectedas separate data files for the mass range 325-1400 m/z while operatingthe instrument at positive polarity. This procedure was performedsequentially to obtain constituent mass data for samples drawn from eachRP-HPLC fraction.

Spectral “ion maps” were generated from the TICs acquired for eachfraction. The maps obtained from corresponding fractions of peptideseluted from different HLA-B15 molecules were aligned (FIG. 13A), andions of interest for NanoES-MS/MS were located. The ion maps weretypically compared following baseline subtraction and smoothing.Putative ligand matches or, in the case of B*1512, mismatches among theions were identified through a combination of data centroiding anddirect visual assessment. Preference was placed upon selectingdoubly-charged, or [M+2H]²⁺, or higher ion forms commonly resulting fromelectrospray ionization for subsequent NanoES-MS/MS since the resultingdaughter ion spectra were richer than those obtained from the collisionof singly-charged, or [M+H]⁺, ions (data not shown).

NanoES-MS/MS was performed by loading into a NanoES capillary tip, asdescribed above, the desired volume of a fraction for which data was tobe acquired. The volume loaded depended upon the relative sample flowrate achieved after opening the capillary tip and how long dataacquisition was intended to proceed. Typically 3-4 μL were loaded at atime to collect MS/MS data for 20-25 mid- to low-intensity ions from agiven fraction. Once loaded, the source head was positioned and thecapillary opened as before. Separate data files were collected for eachion subjected to collisional dissociation.

Daughter ion spectra were generated from the TICs obtained in thismanner for each ion chosen. The specific approach taken to interpetindividual MS/MS spectra varied from ion to ion depending upon thequality of the data sets obtained but adhered to the general rules ofMS/MS fragment interpretation (Roepstorff and Fohlman 1984). The PredictSequence algorithm included as part of the BioMultiView software(BioToolBox package, PE SCIEX) was employed for the de novo sequenceinterpretation, and the sequences deduced were checked for identity withsource proteins in various databases using the PeptideSearch algorithm(European Molecular Biology Laboratory; Mann and Wilm 1994) andperforming advanced BLAST searches (Altschul et al. 1997) against theNational Center for Biotechnology Information (National Institutes ofHealth) databases.

Leu and Ile were indistinguishable unless suggested by Edman data and/orspecific sequence matches, as were Gln and Lys since lysylderivatization prior to fragmentation was not performed. NanoES-MS/MSdata from ions of potentially overlapping peptides was aligned toconfirm or refute the presence of shared ligands among different HLA-B15molecules, as shown for one ion confirmed as an overlapping peptideacross B*1501, B*1503, and B*1508 in FIG. 13, (B and C). Reproducibilityof the protocol in its entirety is demonstrated in FIG. 14. To establisha numerical description (N_(sum)/C_(sum)) comparing ligand N- andC-regional occupancies for each allotype, N and C values for the fourligand positions at either terminus were determined by summingoccurrence frequencies (using an arbitrarily-defined baseline of 10%);N_(sum) was subsequently calculated from the four N values, and C_(sum)was calculated from the four C values.

Peptides from HLA-B15 molecules were subjected to pooled Edmansequencing as well as more extensive examinations, including fractionalEdman sequencing and mass spectrometric characterization of individualligands. This was done to: (i) confirm the production/purificationmethods employed; and (ii) evaluate the relative nature and complexityof the peptides contained in extracts of naturally presented ligands.

Upon extracting peptides from each of six different B15 molecules,pooled Edman sequencing was performed. This was done both to validateresults from the extraction of sHLA ligands with the techniquespreviously employed by others (B*1501 and B*1508, Falk et al. 1995;Barber et al. 1997), and to obtain novel motifs from the molecules thathad not been previously examined (B*1503, B*1510, B*1512, and B*1518)for providing “traditional” points of reference.

Overall, the B*1501 motif (FIG. 15) was in agreement with the dominantP2 and P9 anchors (Gln and Tyr/Phe, respectively) previously defined(Falk et al. 1995; Barber et al. 1996; Barber et al. 1997). This resultdemonstrates that the peptides extracted from sHLA-B*1501 complexes wereidentical to those extracted by others from natural membrane-boundmolecules. However, differences in the whole pool sequencing dataarising from sHLA purified by BBM.1 (FIG. 15B) versus W6/32 (FIG. 15A)indicated that a greater number of peptides than originally realizedshould actually contribute to the consensus B*1501 motif.

First, while Gln and other residues including Leu, Met, and Val havebeen previously reported at P2 using W6/32 to isolate complexes, thealiphatic side chain Pro was strongly detected at P2 in theBBM.1-purified B*1501 motif as well. Though not employed by other groupspursuing similar studies, the β₂m-specific MAb BBM.1 was initiallychosen here to avoid biases potentially imposed upon the class I heavychain by bound peptides (Bluestone et al. 1992; Catipovic et al. 1992;Solheim et al. 1993). Of interest was that Pro has not been reportedbefore as a strong or even weak P2 anchor in the B*1501 peptide motif.It has been suggested that B-locus allotypes that present peptides withPro at P2 demonstrate a shallower B-pocket within their binding groovesthan does B*1501, which exhibits a Ser at α-chain position 67 ratherthan a more constricting residue such as Phe (Barber et al. 1997).

This data suggests the possibility that Pro binds amicably within thisdeeper pocket but perhaps induces an altered heavy chain conformationthat negatively biases purification of complexes by the W6/32 MAbtypically used. The observation of a P2 Pro occupying a pocket withsuboptimal physical complementarity is corroborated by a similaroccurrence among peptides bound by the murine class I molecule L^(d)(Corr et al. 1992; Balendiran et al. 1997). Purification methodologyserves, therefore, as a factor in allele-specific motif predictions, andthe whole pool sequencing with peptides extracted from both BBM.1 andW6/32-purified B*1501 complexes demonstrated that a strong Pro anchor atP2 is antibody dependent.

The pooled Edman motifs obtained for W6/32-purified molecules divergentfrom B*1501 are shown in FIG. 16. Like B*1501, each of the motifsreflected a nonameric consensus with distinct P2 and P9 anchors andinternal auxiliary anchor preferences. The B*1508 motif, described byanother group while its preparation was in progress during thedevelopment of this invention (Barber et al. 1997), was consistentbetween the two laboratories; it demonstrated a preference for the smallside chains Pro and Ala at P2 and aromatic residues Tyr and Phe at P9.The B*1512 motif appeared nearly identical to that obtained from B*1501;by extension, considering that B*1519 differs from B*1512 in α₃, whichdoes not contribute to the peptide binding groove, it is predicted thatB*1519 would bear the same motif as B*1501 and B*1512.

B*1503 diverges somewhat from the other three molecules presented abovein showing a distinct preference for ligands with a neutral, polar Glnor positively-charged Lys as the P2 anchor; the aliphatic Met wasevident here as well, though to a lesser degree than noted for thehydrophilic Gln and Lys residues (Prilliman et al. 1999). Like B*1501,B*1508, and B*1512 however, aromatic residues Tyr and Phe defined ahydrophobic P9 anchor. The only other class I molecules with motifswhose definitions thus far indicate a Lys at P2 are B*3902 (Falk et al.1995) and B*4801 (Martinez-Naves et al. 1997), both of whichstructurally bear B-pockets identical to B*1503 except for a single L→Tor L→E substitution, respectively, at the α₂ helical residue 163(Chelvanayagam 1996). The B-pocket of B*1503 is indistinguishable fromthat of B*4802 (Chelvanayagam 1996), whose motif remains undeterminedbut is likely to follow suit with those of B*1503 and these othermolecules at the second ligand position. An assortment of polar,charged, and hydrophobic residues is evident at P3 of the B*1503 motif.

The B*1510 motif demonstrated a strict preference for ligands bearing abasic, hydrophilic His as a P2 anchor. A hydrophobic P9 anchor wasdescribed by residues including Leu and Phe. The B*1510 motif stronglyresembled that previously defined for B*1509, which exhibits nearlyidentical anchor preferences with His at P2 and Leu, Phe, and Met at P9(Barber et al. 1997). B*1510 and B*1509 differ structurally only by asubstitution of N→D in α₂ at the α-sheet floor position 114, which takespart in forming several specificity pockets within the peptide bindinggroove (FIG. 3).

By extrapolation from its structural neighbors, it was assumed thatB*1518 would have for its motif a P2 anchor of His (as seen for B*1510and B*1509) and a P9 of Tyr and Phe (as seen with B*1501, B*1503,B*1508, and B*1512). B*1518 differs from B*1510 solely at position 116;two other HLA-B molecules that differ exclusively at this position areB*3501 and B*3503: they differ by a S→F substitution here, which wouldsterically mimic the substitution between B*1518 and B*1510 and conferB*1510-like P9 preferences (Steinle et al. 1995; Kubo et al. 1998).Based upon this, and the fact that the P9 environments of B*1518/B*3501and B*1510/B*3503 are similar (Chelvanayagam 1996), it was firstpredicted, and then confirmed following pooled sequencing, that B*1518would bear the “hybrid motif” described.

Pooled Edman sequencing data therefore demonstrates that (i) thepeptides extracted from sHLA complexes produced according to themethodology of the present invention are consistent with thosepreviously extracted from native, cell surface-expressed complexes, and(ii) nonameric ligand lengths with anchor residues at P2 and P9characteristic to specific polymorphisms are preferred. As forfunctional implications, the major anchors would predict natural ligandoverlaps with B*1501 by B*1503 and B*1512.

Since class I peptide pools consist of thousands of different ligands,an early investigation during development of the analysis strategy andmethods disclosed herein was to next fractionate and then Edman sequencethe peptides extracted from one of the molecules. BBM.1-purified B*1501(Prilliman, et al. 1997), the first soluble molecule produced by anon-repeatable precursor methodology to the fully repeatable andcharacterized methodology of the present invention, was initiallyexamined to explore the general diversity around a pooled motif(Prilliman et al. 1998).

Edman sequencing of peptide-containing fractions collected from theRP-HPLC gradient shown and described in FIG. 17 supported the existenceof peptides up to 12 residues long and revealed significant positionaldiversity among the peptides isolated from B*1501. This positionaldiversity was illustrated in 16 representative fractions (FIG. 18).Assessment of dominant, strong, or weak amino acid residues present atthe various positions indicated that the dominant anchors previouslydefined by whole pool sequencing did not necessarily predominate infractions of the peptide pool (FIG. 19). In fractions 15 and 31 thedominant P2 Gln was replaced by a dominant Ala and a dominant Lysrespectively, while the pooled motif Tyr fell below residues such as Hisand Lys at P9 in the same fractions. While variations on the consensusmotif were prevalent at P2, this was not solely restricted to the Ntermini of bound peptides as shown in FIG. 18.

Among the 16 fractions studied, 12 demonstrated weak sequence yields outto 12 cycles of degradation; in nine of these fractions, P12 wasoccupied by the charged or polar residues Glu, Arg, Lys, Ser, His, orGln. Additionally, the presence of numerous decamers within the B*1501peptide population was suggested in that 11 of the fractions sequencedexhibited a typical P9 residue, Tyr or Phe, at P10. For example, in FIG.19 the strong Phe presence at P10 in fraction 28 suggests that P10serves as an anchor for a decamer(s) present within the fraction. Theshifting of a P9 anchor preference to P10 is consistent with the P10occupancies of individual decamers formerly characterized from B*1501peptide pools (Falk et al. 1995; Barber et al. 1997). Although residuesrepresenting P9 anchors were seen at P10 and P12, amino acids unique insuch longer ligands were also detected, suggesting that the shifting ofa previously-reported P9 anchor is not the only means by which longerB*1501 peptides are bound within the peptide binding groove (Collins etal. 1994).

During the course of examining ligands from other sHLA molecules, whichwere purified alternatively with MAb W6/32, aliquots of RP-HPLCfractions from some were also subjected to Edman sequencing on occasion.The results from these random samplings of B*1501, B*1508, B*1503, andB*1510 fractions are summarized for major characteristics in Table 3.The characteristics recorded included preferences other than those seenin the pooled motifs at traditional anchor positions P2 and P9 andwhether signal levels indicated the presence or not of residues beyondthe nine cycles of degradation typically observed. Of additionalinterest, no Lys was observed among Edman sequenced fractions as anadditional preference at P2 for B*1501 purified with W6/32, a findingcontrary to the P2 preferences among fractions of BBM.1-purifiedmaterial which lends further support to the argument for MAb bias, asdiscussed earlier, existing in complex purification.

In summary, the fractionation of B*1501 peptides prior to Edman analysisresulted in amino acid sequence data demonstrating that the componentsof a peptide pool can vary considerably from the overall motif(Prilliman et al. 1998). These data, as well as results obtained fromother B15 molecules, suggested that peptides bound by the variousmolecules include: (i) species which are either longer or shorter thanthe nonameric size typically indicated by pool sequencing alone; and(ii) species that exhibit primary sequences different from thosepredicted by pooled sequencing. The additional data provided by furtherexploration of fractionated extracts from several of the B15 moleculesin this manner expands the molecules with the potential for presentingB*1501-overlapping ligands to include not only B*1503 and B*1512 butB*1508 and B*1510 as well. Such diversity among fractions indicated thatcharacterization of individual ligands would provide information notavailable in motifs.

Individual peptides from B*1501, B*1503, B*1508, B*1510, and B*1512 werecomparatively examined to investigate whether the added flexibilityobserved through Edman degradation of RP-HPLC fractions would allow fornatural ligand overlaps to occur across their respective polymorphisms.

More than 400 individual ligands extracted from the five distinctHLA-B15 allotypes were characterized according to the methodology of thepresent invention. The ligands characterized here were from ion mapmasses found in multiple B15 allotypes as demonstrated in FIG. 13A.Selected ions were then dissociated by NanoES-MS/MS, and the resultingfragment information was compared and interpreted, as describedhereinabove, to determine if the ions represented sequence-identical ormerely mass-identical ligand matches.

Individual peptide ligands characterized from the five B15 allotypes arelisted in Tables A-E. The number of ligands for which either complete orpartial sequences were obtained here was as follows: B*1501=126,B*1503=74, B*1508=96, B*1510=123, and B*1512=30. While the pooled motifsof peptides extracted respectively from the five molecules describednonamers with various P2 and P9 dominant anchors and P3 auxiliary anchorpreferences (FIGS. 15 and 16), the single peptide sequences ranged from7 to 12 amino acids in length and demonstrated (i) greater heterogeneityat their N-terminal/proximal regions than their C termini, and (ii)varying degrees of observed ligand overlap, both of which will beexamined in the subsequent sections of this chapter.

In terms of length heterogeneity, the endogenous peptides eluted fromB*1501, B*1503, B*1508, B*1510, and B*1512 varied in length from 7 to 12amino acids as shown (FIG. 20). An overall length breakdown of thepeptides listed in Tables A-E demonstrates that approximately 6% areheptamers, 21% are octamers, 50% are nonamers, 19% are decamers, 3% areundecamers, and 1% are dodecamers. Further emerging from the lengthcharacterization of individual ligands is the observation that peptidesbound by each of the B15 molecules spanned ranges of 5 to 6 amino acidsin length. For example, peptides eluted from B*1501, B*1510, and B*1512were 7-11 amino acids in length, while those from B*1503 and B*1508 were7-12 amino acids in length.

Coupling this length variability with the likewise varying degrees ofregional sequence heterogeneity (which will be discussed) leaves only23% of the endogenously loaded peptides characterized in Tables A-E as“ideal nonamers” with both P2 and P9 anchors in concordance with thedominant or strong preferences of the pooled Edman motifs from theirrespective source molecules. This finding is of principal significancein that a majority (77%) of potential ligands for any of these HLA-B15molecules would therefore be overlooked if the length and sequenceconstraints of their pooled motifs were utilized as the primary criteriain searching for potential epitopes specific to them.

Examples of ligands from this study with homology to stretches of knownproteins are shown in Table 4. The peptides yielding 100% identicalBLAST database hits were grouped into seven categories, which weredefined here according to the common natures of their potential sourceproteins: HLA ligands, replication/transcription/translation ligands,biosynthetic/degradative modification ligands, signalling/modulatoryligands, transporter/chaperone ligands, structural/cytokinesis ligands,and unknown function ligands. Aside from the HLA heavy chain-derivedligands, most appear to be derived from cytoplasmic or nuclear proteins,which illustrates that the typical endogenous pathway is involved ingenerating the majority of the class I-loaded peptides characterized(York and Rock 1996).

Of the 44 peptide sequences listed in Table 4, it is noteworthy thatoverlaps across other HLA-B15 molecules are evident within our datacollection. The B*1510 tapasin₃₅₄₋₃₆₂ ligand HHSDGSVSL (SEQ ID NO:51),as well as both THTQPGVQL (SEQ ID NO:54) from septin 2 homolog₇₀₋₇₈ andSHANSAVVL (SEQ ID NO:55) from β-adaptin₂₄₉₋₂₅₇, have also been sequencedfrom B*1509 extracts (Barber et al. 1997), and the B*1501/B*1508/B*1512ubiquitin-protein ligase₈₃₋₉₁-derived ligand ILGPPGSVY (SEQ ID NO:41)was characterized from endogenously bound B*1502 peptides (Barber et al.1997). The eIF3-p66₆₁₋₆₉ nonamer SQFGGGSQY (SEQ ID NO:29) (Falk et al.1995; Barber et al. 1996) was found here within B*1501, B*1503, B*1508,and B*1512 extracts. The decamer YMIDPSGVSY (SEQ ID NO:42), which ishomologous to proteasome subunit C8₁₅₀₋₁₅₉, was also previouslydescribed as a ligand for B*1502 (Barber et al. 1997), B*1508 (Barber etal. 1997), and B*4601 (Barber et al. 1996); it was found here presentedby B*1501, B*1508, and B*1512. Some of the specific overlapping ligandsidentified in this study therefore overlap in antigen presentation withthe HLA-B15 allotypes characterized by others.

Given the length heterogeneity observed among the ligands collectivelycharacterized from B*1501, B*1503, B*1508, B*1510, and B*1512, analysisof peptide ligand primary structures proceeded through two separatealignments (N- and C-terminal) for each HLA-B15 allotype. Thefrequencies with which specific side chains occurred at (i) the Nterminus and first three residues internal from it, and (ii) the Cterminus and the first three residues internal to it were tabulated. Asgraphically summarized throughout parts A and B, respectively, of FIGS.21-25, the salient features of these ligand regions and how theycorrelated with the structures of the molecules from which they wereextracted can be examined.

The N-terminal/proximal regions for ligands from each of B*1501 (FIG.21A), B*1503 (FIG. 22A), B*1508 (FIG. 23A), B*1510 (FIG. 24A), andB*1512 (FIG. 25A) clearly demonstrated acceptance of a variety of aminoacid side chains, particularly at P3 and P4, by the portions of thebinding groove assumed to interact with ligands at the designatedpositions. With the exception of B*1512 ligands, which were obtainedfrom both a smaller and more biased collection of ions (see FIG. 25legend), higher points in each of the graphs occur for certain sidechains indicated along the P1 and, to a greater extent, P2 data lines,which represent the first and second positions, respectively, of thecharacterized ligands.

The results for P1 obtained from the HLA-B15 ligands characterized areof interest since the analysis of Edman sequencing data depends uponcomparing relative increases between cycles and is therefore unreliablefor making side chain determinations at this first position, especiallywhen complex mixtures of peptides are examined (Stevanovic and Jung1993). All five B15 allotypes demonstrated a number of side chains atP1, with preferences for residues including Ala, Leu/Ile, Gly, Ser, Thr,and Tyr observed in varying degrees among them (FIGS. 21-25, A).Overall, P1 appeared to be occupied in a majority of ligands byaliphatic amino acids.

Though the pooled motifs of B*1501 and B*1512 (FIGS. 15 and 16) as wellas the spectral ion maps obtained from their RP-HPLC fractions (data notshown) were virtually identical, the substitution difference between thetwo molecules at A-pocket residue 167 suggested that ligands bound bythese molecules might differ at P1. Performing NanoES-MS/MS upon ahandful of ions, which appeared to be exclusive to B*1512, confirmed thepresence of several ligands presented by B*1512 but not B*1501 (Table5). Of the 16 sequenced, seven indicated His, two indicated Arg, and oneindicated Lys at P1. In comparison, only a single B*1501 peptide eachpresented with His, Arg, or Lys at P1 out of 126 ligands characterized(Table A). An explanation for the existence of this subset ofB*1512-restricted ligands with positively-charged N-termini could lie inthe W→G substitution observed between B*1501 and B*1512 at α₂ position167, which might sterically enhance the influence by the adjacent acidicresidue at position 166 (Glu in B*1501 and Asp in B*1512) of B*1512 uponP1 in the A-pocket. Comparing individual ligands between B*1512 andB*1501 supports the notion that the polymorphism segregating them willconfer distinct yet subtle effects upon peptide binding by otherallotypes differing in this manner.

P2, which has been considered to act as a primary anchor for peptideligands among most class I molecules described to date as based uponpooled Edman motifs, is classically accepted to associate with theB-pocket of the peptide binding groove. In terms of the motifs derivedhere by pooled sequencing (FIGS. 15 and 16) and the motifs previouslyestablished for other B15 family allotypes (Falk et al. 1995; Barber etal. 1996; Barber et al. 1997), a Gln at P2 is common to B*1501, B*1502,B*1503, B*1512, and B*1513 (Table 6). Three alleles, B*1502, B*1513, andB*1508, have a Pro at P2, while the lack of a strong Pro at P2 in bothB*1501 and B*1503 corresponds to polymorphism at heavy chain positions63 and 67. For example, B*1501 appears to lose the propensity for Pro atP2 due to polymorphism at position 63, while B*1508 appears to lose aGln at P2 resulting from polymorphism at 67. Thus, comparisons withinthe B15 family highlight how substitutions at positions 63 and 67 of theclass I heavy chain α₁ helix appear to confer differential interactionwith P2 of the peptide ligand.

While residue 63 modulates the size/conformation of P2, it can be seenthat residues 24 and 45 influence the P2 charge nature propensities. Acomparison of B*1501 and B*1503 illustrates how polymorphisms atpositions 24 and 45 of the B-pocket influence P2 preferences in thismanner; B*1503 is one of four B15 alleles with known motifs bearing apositively charged P2. Allotypes B*1509, B*1510, and B*1518 recognize apositively charged His at P2 and have the same residues at 24 and 45 asB*1503, but the differences at positions 63 and 67, which separateB*1503 from the other three molecules, again modulate the contour of P2such that different positively charged P2 residues fit respectively intothe B*1503 and B*1509/B*1510/B*1518 B-pocket categories.

It has previously been proposed that polymorphisms in the α₁ helixprompt major changes in the repertoires of peptides bound by allotypesdiffering in this region (Barber et al. 1997). That any region, helicalor sheet, of α₁ would influence peptide P2 preferences more than α₂ isof little surprise though since 14 of the 18 residues forming theB-pocket belong to the α₁ domain (Table 6). A comparison of 12 known B15motifs in the B-pocket suggests more refined rules for α₁ in general,whereby polymorphisms in the helices sculpt the conformation and size ofthe amino acids that can fit into the peptide binding groove's B-pocket.Further analysis of the B15 motifs at P2 suggests that polymorphismslining the floor of the groove tend to regulate the hydrophobic and/orcharged nature of the residues at P2 of bound ligands. Perhaps in thisway the walls and floor of the binding groove work in concert: theα-helical residues sterically control which amino acids can fit, whilethe β-sheet residues act to attract or repel particular side chainsbased on chemical compatibility within the ligand binding groove.

The interactions described for P2 here are, however, more relaxed thanpreviously thought. For a majority of the allotypes shown in Table 6,three or more different side chains are observed by pooled Edmansequencing as dominant or strong B-pocket residents and/or the B-pocketsof the molecules demonstrate abilities to naturally accommodatealternative P2 residues (Table 3 and FIGS. 21-25, A). Of specificinterest, although Met appears in the B*1503 pooled motif as a strong P2anchor residue (FIG. 16), only two of the peptides characterized forthis allotype bear Met at P2 (FIG. 22A), while other residues includingGly, Pro, Ala, and Asn occur more often than Met at P2 among B*1503ligands. The fact that Met appears in the pooled motif but fails todemonstrate a strong presence among the individual peptides indicatesthat disparate concentrations of ligands within extracts may skew pooledEdman sequencing results so as to be misleading.

Pro and Ala likewise appear with frequencies comparable to or exceedingthose of the W6/32-purified pooled motif residues for B*1501 (FIG. 21A),and B*1508 ligands illustrate P2 inclinations for a rich array of sidechains in addition to the motif-prescribed residues Pro and Ala whichinclude Gly, Val, Met, Leu/Ile, Ser, Thr, and Gln/Lys. Similar varietyis observed within the limited B*1512 ligand data set (FIG. 25A). Incontrast, the B-pocket composition for B*1510 indicates His as the soledominant/strong P2 occupant (Table 6), and among individual ligandscharacterized from B*1510 His is noted at a markedly higher degree thanare alternative residues (FIG. 24A). However, amino acids including notonly the positively charged Arg but to a greater extent Gly, Ala, Val,Leu/Ile, and Gln/Lys occur at P2 of some peptides are also characterizedat P2 from this allotype. Thus the majority of HLA-B15 moleculesdemonstrate elastic N-proximal occupancies.

In comparison with the findings at the N-terminal/proximal regions, theC termini of ligands from each of B*1501 (FIG. 21B), B*1503 (FIG. 22B),B*1508 (FIG. 23B), B*1510 (FIG. 24B), and B*1512 (FIG. 25B) demonstrateda stricter acceptance of amino acid side chains. C-proximal ligandresidues also revealed the existence of more distinct side chaintendencies.

For allotypes B*1501, B*1503, B*1508, and B*1512 a dominant C terminuswas especially prominent among the ligands characterized from them,while B*1510 exhibited a P2 anchor nearly as strong as its primaryC-terminal residue preference (FIG. 24, A and B). The aromatic residuesPhe and, even more prominently, Tyr occupied the C-terminal positions ofmost peptides bound by the first four B15 molecules, which appeared toagree with P9 of their respective motifs (FIGS. 15 and 16). The bulk ofB*1510 ligands demonstrated Leu/Ile at their C termini; other occupantsat this position included Phe and Val, an interesting observation inthat more B*1510 peptides presented with Val, which is not included ineither the pooled (FIG. 16) or fractional (Table 3) Edman motifsexamined from this allotype, than Phe, which is identified as a strongP9 occupant by pooled sequencing. Such is the likely result of disparatepeptide concentrations affecting the pooled Edman sequencing results asmentioned previously. Another factor includes the diminishing picomoleyields per successive cycle of Edman degradation; this leads toprogressively higher background signals and thus negatively affectssensitivity in examining the C-terminal/proximal regions of peptides(Stevanovic and Jung 1993).

The overwhelming conservation at the C termini of individual ligands(FIGS. 21-25, B) indicates that the C terminus acts as a dominant anchorfor peptide ligands. P9 of pooled Edman motifs has classically beenaccepted to associate with the F-pocket of the peptide binding groove.C-terminal anchoring is observed here regardless of length heterogeneity(FIG. 20 and Tables A-E). For the majority (91%) of peptides greaterthan 9 residues long, this observation agrees with evidence that longerpeptide ligands bulge centrally outward from the peptide binding groove.Sequencing individual ligands supports a concept that the C terminus ofa ligand plays a dominant role as an anchor within the class I bindinggroove for the HLA-B15 allotypes examined.

With regard to pooled sequence motifs for HLA-B15 allotypes, all 12molecules demonstrate chemical homogeneity at P9, with dominant/strongoccupancies by hydrophobic residues (Table 7). The eight differentF-pockets structurally represented among these allotypes showpreferences for Tyr, Phe, Met, Leu, and Trp according to threefunctional group categories. This is in marked contrast with theB-pockets, for which eight different B-pockets among the same allotypescomprised seven distinct functional groups encompassing a mixture ofboth hydrophobic and hydrophilic side chains (Table 6).

It is interesting that, of these HLA-B15 molecules, nine have F-pocketfunctionality in the same category (B*1501, B*1502, B*1503, B*1508,B*1512, B*1516, B*1517, B*1518, and B*4601), with preferences for Tyr,Phe, and/or Met, despite the fact that they exhibit amino acidsubstitutions at nine different positions throughout the α₁ helix and β₂sheets. This redundancy demonstrates that, contrary to what was seenamong structural residues affecting the B-pocket, the α₁ helicalpolymorphism(s) shown for allotypes in the first category of Table 7 donot necessarily play a defined role in sculpting either the conformationor size preferences of ligands in this region of the peptide bindinggroove.

While the Edman-derived motifs for the B15 allotypes shown in FIGS. 15and 16 clearly indicated P2 and P9 primary anchors and suggest anassortment of preferences at both P3 and P4, they fail to sufficientlycapture the trends for auxiliary anchoring at the C-proximal regions ofendogenously bound ligands which were perceptible throughout theindividual peptide sequences. Additional preferences that likely serveas auxiliary anchors were evident at the C-proximal positions C⁻¹, C⁻²,and C⁻³ in the cases of nonamers, octamers, and heptamers, as well as inligands longer than nonamers. A review of the positional frequenciesobserved among the HLA-B15 peptides shows that amino acids such as Val,Leu/Ile, Ser, Thr, and Gln/Lys tended to predominate at nearly all threeof the C-proximal positions of ligands presented by B*1501, B*1503,B*1508, B*1510, and B*1512 (FIGS. 21-25, B). Of these residues, thehydrophilic, hydroxyl-containing Ser and Thr were especially frequentamong these positions; nearly half (40%) of the ligands listed in TablesA-E, bear Ser and/or Thr at the designated C-proximal positions. Ingeneral, Glu occupied C⁻¹ and Gly occupied C⁻³ to some extent among allfive allotypes.

Val (C⁻¹) and Pro (C⁻²) were especially prominent C-proximal residuesobserved among the B*1510 ligands; the overriding presence of Pro, whichdistinguished this region of B*1510-derived peptides from those of theother allotypes, can likely be attributed to steric influences imposedby the Tyr at α₂ position 116 in B*1510, which additionally interactswith the C- and E-pockets of the peptide binding groove (FIG. 3).Further distinguishing several B*1510 ligands from but rare occurrencesamong B*1501, B*1503, B*1508, and B*1512, Pro frequently appeared aswell in various C-proximal sequence combinations with Ala or Val (Table8).

The amino acid residues characterized from each of the five HLA-B15allotypes with occupancy rates of at least 10% for the first four(N-terminal/proximal) and last four (C-terminal/proximal) positionsamong ligands, respectively, are condensed in Tables 9-13. Presentingthe data already discussed in this manner effectively emphasizesC-terminal dominance and N-proximal flexibility. By comparison, the dataillustrates the limitations of pooled Edman motifs in being able toadequately reflect a consensus of the individual peptides containedwithin a given ligand extract. The N_(sum)/C_(sum) quotients obtained asdescribed for Tables 9-13 were less than 1.00 in the cases of allallotypes, thus providing a more fixed description to theC-terminal/proximal region (gray) as a whole with respect to theN-terminal/proximal region (black).

Among the N-regional position ligand residues occurring at >10%, nothingappears to prominently stand out at P3 and P4 although assignments weremade to these positions via Edman sequencing. The occupancies that wereobserved were not necessarily captured by the motifs; a specificillustration of this is Ala (19.51%) at P3 of B*1510 (Table 12 and FIG.16). This trend appeared likewise applicable at the P2 anchor, wherewith the exceptions of B*1508 and B*1510, occupants at this positionamong >10% of ligands from each of the remaining allotypes includedadditional side chains (for example, Ala at P2 in both B*1501 andB*1512) not accounted for by pooled sequencing. The C termini of eachallotype are comprised of two amino acid specificities as shown by morethan 80% of characterized peptides in all cases (Tables 9-13). Insummary, comparing observed N- and C-regional occupancies among thecharacterized ligands underscores the flexibility of N-proximal versusthe dominance of C-terminal preferences among the B*1501, B*1503,B*1508, B*1510, and B*1512 binding grooves.

A total of 40 specific ligands among the 449 characterized here (TablesA-E) overlapped across the peptide binding grooves of B*1501, B*1503,B*1508, and/or B*1512 (Table 14); as previously discussed from theinformation in Table 4, some of the overlapping ligands likewisecoincided with ligands characterized by others from additional HLA-B15allotypes including B*1502 and B*4601. Length variations among theoverlapping ligands identified tended to mimic those observed among theentire set of ligands characterized, formerly discussed and illustratedin FIG. 18. Only seven overlapping ligands were longer than 9 aminoacids in length (4 decamers and 3 undecamers), while 16 fell short of 9residues long (6 heptamers and 10 octamers); less than 50% of thesuccessful overlaps were therefore nonameric.

Throughout the mapping and sequencing approach that was developed andexecuted as outlined earlier (FIG. 6), an extensive comparison was firstconducted upon ions occupying RP-HPLC fractions 6-19 from separations asshown in FIG. 11 of ˜400 μg of peptides from each of B*1501, B*1503,B*1508, and B*1510, the first four B15 molecules prepared in the courseof this study. From this, 21 peptide overlaps across B*1508 and B*1501were defined. Similarly, eight ligands overlapping B*1501 and B*1503were identified, and four ligands were found to overlap across B*1508,B*1503, and B*1501. A conservative estimate, based upon past examinationof B*1501 ligands, is that the ion maps for each of the B15 allotypesrepresented at least 2,000 individual peptides per molecule (Prillimanet al. 1997), yet B*1510 was not observed to share ligand overlaps withB*1508, B*15011, or B*1503 (Table 14).

The sequence data indicates that overlapping ligands bind acrossdivergent B*1508, B*1501, and B*1503 binding grooves but not B*1510.This pattern likewise accentuates an apparently dominant role forC-terminal anchors in natural peptide binding as discussed previously.FIG. 26 depicts, in the context of the class I peptide binding cleft,the locations of polymorphisms that individuate B*1508, B*1501, B*1503,and B*1510 and highlights the anchoring residues for the peptideoverlaps according to the N-proximal and C-terminal specificities oftheir respective presenting molecule's motif (FIGS. 15 and 16). Boldingthe amino acids of these overlapping ligands, which are in agreementwith the traditional pooled motifs, underscores the trend whereby aC-terminal anchor sequence is conserved within overlaps while theN-proximal anchor is considerably more flexible in its location and/orsequence. A lack of overlaps with B*1510 could potentially be explainedby the S→Y substitution observed between this allotype and the otherthree at α₂ position 116. Thus, the conserved C-terminal anchors thatfacilitate the occurrence of B*1508, B*1501, and B*1503 overlaps fail topreferentially interact with the B*1510 C-terminal specificity pockets.

A further example provided here of how C-proximal auxiliary anchorsmight positively impact endogenous ligand binding is that eight of thepeptides overlapping both the B*1508 and B*1501 antigen binding groovesbear Thr at C⁻¹, C⁻², or C⁻³, and in four cases the peptides that bindB*1508/B*1501 or B*1508/B*1501/B*1503 are heptamers with Thr occupyingP7, their C-terminal positions (FIG. 26). The prominent role of Thr as aC-terminal/proximal auxiliary anchor is dramatically illustrated by theB*1508/B*1501/B*1503 overlapping heptamer CPLSCFT (SEQ ID NO:60), whereThr provides a C-terminal anchor for this ligand not evident in thepooled motifs of the three allotypes.

Distilling the data from the overlapping ligands among B*1501, B*1503,and B*1508 suggests a model for endogenous ligand binding wherebypeptides are first anchored or held in the class I binding groove bytheir C termini. In order for a given peptide to remain stably fastenedin the groove for successful trimer assembly and subsequent export fromthe cell, it is observed that following rigid anchoring at the Cterminus as described, a ligand must be subsequently tethered into theclass I antigen binding cleft at a more variably defined N-proximalposition. Such is the case for peptide ligand NQZHGSAEY (SEQ ID NO:138),a nonamer that overlaps across B*1508, B*1501, and B*1503 (FIG. 24).According to this model (FIG. 27), a C-terminal Tyr securely anchorsNQZHGSAEY (SEQ ID NO:138) into all three B15 allotypes, while a Gln atP2 anchors the peptide into B*1501 and B*1503 and a Gln/Lys (most likelya Lys based upon both motif assignments and fractional Edman sequencingdata) at P3 provides additional anchoring for B*1501 and serves as thesole N-proximal anchor for B*1508. This model appears clearly applicableto at least 75% of the ligands presented in FIG. 26; for those peptidesto which it does not evidently apply, the possible anchoring modesremain open to further speculation at the level of individual ligands.

For example, the B*1501/B*1503 overlap AQFASGAGZ (SEQ ID NO:135) (FIG.26) may instead be additively stabilized through the N-proximal anchorsindicated at P2 and P3 as well as at the N-terminal position, since Alademonstrated significant P1 occupancy among both B*1501 and B*1503ligands, as previously shown (FIGS. 21 and 22, A). Additionally, thefour heptameric overlaps that were observed across B*1508/B*1501/B*1503,which terminate in Thr, could lie within the peptide binding groove suchthat they are anchored N-terminally/proximally and their C terminiinteract with the C-proximal regions of the groove, which havedemonstrated preferences for Thr; these ligands might therefore fail toextend into the F-pocket. As compared with C-terminal sequences, bothlength and N-proximal specificity characteristics of ligands generallyplay secondary roles in the natural binding of B15 peptide epitopes.

Further stemming from the data obtained by comparatively examiningB*1501, B*1503, B*1508, and B*1510 ligands, differential interactionswith the chaperone tapasin specifically influence the loading ofpeptides into HLA-B15 molecules. Tapasin, an MHC-encoded chaperonediscussed hereinabove (Herberg et al. 1998), is a recently-discovered 48kDa transmembrane glycoprotein resident to the ER that directlyinteracts with both calreticulin and empty α-chain/β₂m dimers to form a“loading complex” linked to TAP1/TAP2 (Sadasivan et al. 1996; GrandeaIII et al. 1997; Li et al. 1999). Tapasin is not a requirement forligand loading via the typical endogenous processing pathway (Lewis etal. 1998; Peh et al. 1998), and aside from its proposed role in servingas a bridge between a class I dimer and the peptide transporter untilrelease of mature trimers upon peptide binding, the exact role oftapasin during class I assembly is unknown (Pamer and Cresswell 1998).Interactions between nascent class I molecules and TAP1/TAP2 have,however, been shown to be influenced either directly or indirectly by α₃and positions 116 and 156 of α₂ (Suh et al. 1999; Kulig et al. 1998;Neisig et al. 1996).

Of specific interest, past analysis of divergent HLA-B35 molecules hasindicated that allotypes bearing an aromatic amino acid (Phe or Tyr) atposition 116 interacted with TAP1/TAP2; allotypes bearing a Sersubstitution at this site failed to demonstrate the interaction (Neisiget al. 1996). Likewise, data subsequently obtained for the sHLAtransfectants utilized here according to established immunoprecipitationprotocols (for example, Harris et al. 1998) indicates that although allfour allotypes associate with calreticulin, B*1501, B*1503, and B*1508do not associate with tapasin (and therefore not with TAP1/TAP2) whereasB*1510 does. Membrane-bound forms of B*1501 and B*1516 have previouslybeen shown by others to not associate with TAP1/TAP2 (Neisig et al.1996; de la Salle et al. 1997), demonstrating that results obtained fromthe sHLA transfects are in concordance with those of native molecules.

Though functionally divergent according to its pooled motif (FIG. 16)and the majority of peptides that it binds, B*1510 is capable ofaccommodating ligands with the properties favored by the B*1501, B*1503,and B*1508 binding grooves. Data both from individual ligands (Table D)and fractional Edman sequencing (Table 3) indicate that Tyr can occupythe C-terminal position, and specific examples in Table 4, including thespleen mitotic checkpoint BUB3₅₃₋₆₀ octamer YQHTGAVL (SEQ ID NO:32) andthe splicing factor U2AF large chain₁₇₉₋₁₈₇ nonamer TQAPGNPVL (SEQ IDNO:37), attest to B-pocket flexibility. It is intriguing that among thepeptides bound by B*1510 is the tapasin₃₅₄₋₃₆₂ nonamer HHSDGSVSL (SEQ IDNO:51); the peptide appears to occupy ligand extracts in a high copynumber, as qualitatively based upon relative mass spectrometric ionintensities. Given this, as well as considering potential models ofloading complex interactions suggested by others (Neisig et al. 1996;Elliott 1997), it can be extrapolated that a portion of tapasin,analogous to class II-associated invariant chain-derived peptides(Riberdy et al. 1992; Sette et al. 1992), extends into and blocks aregion of the empty class I binding groove until it is displaced by anoptimally-fitting ligand and/or secondary chaperone; this could alsoaccount for the differences in overall P2 flexibility observed betweenB*1510 peptides and those of the other three allotypes. Participating inligand selection by this mechanism would describe a distinct peptideediting role for tapasin and could clarify the inability to detectoverlaps between B*1510 and either B*1501, B*1503, or B*1508.

In addition to the initial search for overlaps across B*1501, B*1503,B*1508, and B*1510, a comparative analysis was performed between the ionmaps of B*1501 and B*1512. As discussed previously hereinabove, such anexamination is primarily important in revealing the presence of ligandsbound by B*1512 but not B*1501 (Table 5). A number of overlappingligands from B*1512, however, were additionally identified (Table 14).Conservative percentages of overlap subsequently observed among each ofthe four molecules from which ligands were characterized and theancestral HLA-B15 allotype, B*1501, were determined as shown anddescribed in Table 15.

As expected from an overview of their nearly identical ion maps, B*1501and B*1512 demonstrated the highest overlap frequency between theallotypes at 70% among ions subjected to NanoES-MS/MS. After this,B*1503 and B*1508 respectively exhibited 14% and 9% overlap frequencies,while as shown earlier B*1510 completely failed to reveal overlaps withB*1501. The trend distinctly illustrates that the polymorphisms whichdistinguish the B*1503, B*1508, B*1510, and B*1512 peptide bindinggrooves from B*1501 are not functionally equivalent in terms of theirimpacts upon class I ligand association. However, it is also evidentthat they do not create concrete barriers to ligand binding. Because therepertoires of peptides bound by the various molecules examined maydiffer from B*1501 at frequencies greater than 80% (B*1508 and B*1503,Table 15) does not mean that they are unable to bind similar orcompletely identical peptides, a concept which has been incompletelyaddressed and occasionally negated by other studies grounded more uponpooled Edman sequencing, analysis of prominent extract constituents, orbinding/reconstitution assays.

In particular and based upon previous research, the overlaps betweenB*1508 and B*1501 defined here would specifically not have beenpredicted (Barber et al. 1997); it may be anticipated by extension thatother molecules differing solely by the polymorphism separating B*1508and B*1501, for example B*1503 and B*1529 (Appendix A), yield similaroverlap frequencies. Likewise, based upon the relatively high overlapfrequency observed between B*1512 and B*1501, the substitutions atpositions 166 and 167 distinguishing them exhibit a similarly subtleeffect between A*2902 and A*2903 (which only differ from one another bythe identical substitutions studied here; Prokupek et al. 1997) viamapping and sequencing of individual ligands. Systematically attemptingto define the limits of overlap existence as conducted, therefore,demonstrates a critical departure from standard approaches which enhancepredicting the abilities of different class I molecules to presentoverlapping ligands.

Comparative analyses of closely related soluble MHC class I moleculesproduced by the recombinant methods described herein, provide a meansfor assessing the functional impact of individual α-chain polymorphisms.The primary impetus for characterizing peptides extracted from class Imolecules is to more precisely understand the influence of structuralpolymorphism upon the presentation of endogenous ligands. This isimportant since a fundamental realization of how naturally processedpeptides bind to both individual and multiple class I allotypes can thenbe translated into protein and/or peptide-based therapies intended toelicit protective CTL responses. Therefore, an accurate interpretationof sequence data from such class I-bound peptides, either individual orpooled, should in turn further the selection of optimal viral andtumor-associated ligands to expedite the development of successfultherapeutic applications.

The extensive examination of HLA-B15 ligands, as described herein,enhances understanding the rules that govern natural class I peptidepresentation and is secondary evidence of the success and usefulness ofthe methodology for producing soluble MHC class I and II moleculesdescribed and claimed herein. By first building upon the traditionalfoundations provided by pooled Edman motifs (FIGS. 15 and 16), the datafrom over 400 individual peptides characterized from B*1501, B*1503,B*1508, B*1510, and B*1512 subsequently indicated that queries forpotential epitopes specific to these allotypes would benefit from beingoptimized in three ways. First, although nonamers represent half of theligand population, the other 50% of peptide epitopes range down to 7 andup to 12 amino acids in length. Second, effective N-proximal anchorrequirements need not be strictly imposed at P2. Third, searches forligands should weigh C-terminal/proximal sequence matches even moreheavily than those of the N-region. The third trend revealed represent,the most substantial of the revised search criteria, since both lengthvariations and lower sensitivity due to the diminishing returns andincreasing backgrounds inherent to successive Edman sequencing cyclescan C-regional motif trends (Stevanovic and Jung 1993). As stressedearlier, examples of this bias were evident here in both: (i) thestronger preference by B*1510 for peptides terminating in Val (absentfrom the pooled motif) rather than Phe (present in the pooled motif)(FIGS. 16 and 24B); and (ii) the inability of motifs to effectivelyreflect C-proximal auxiliary anchors (FIGS. 15; 16; and 21-25, B).

To illustrate the potential consequences of applying the modified searchparameters described, the EBV structural antigen gp85, which hasrecently been implicated using a murine model as a favorable targetagainst which protective CTLs might be generated (Khanna et al. 1999),was examined in the context of B*1501 to identify: (i) nonamericepitopes with motif-prescribed P2 and P9 occupancies; (ii) lengthvariant epitopes with motif-prescribed P2 and P9 occupancies; and (iii)nonameric epitopes with flexible P2 occupancy (Table 16). Since onlythese three categories of ligands were designated, the inquiry was notexhaustive. However, the information extracted showed that, of the 98possible epitopes identified, only the 22% within the first column wouldbe placed under further experimental consideration if pooled motifsalone were applied in the search. This is not to imply that the data inthis category is invalid but that it might be considerably incompletefor later applications. For example, if either of the AMTSKFLMGTY₁₇₂₋₁₈₂(SEQ ID NO:205)(varying by length) or the SAPLEKQLF₁₂₃₋₁₃₁ (SEQ IDNO:246)(varying by P2 occupancy) peptides was demonstrated to elicit amore effective antigen-specific CTL response than any of the nonamersbearing standard motif P2/P9 assignments, this knowledge is pivotal tosubsequent vaccine design; even if the two designated peptides evokedresponses only equivalent to some of the nonamers, their non-motiflength and/or sequences discrepancies could prove superior in conferringthe ability to overlap multiple allotypes in addition to B*1501. This isadvantageous since a vaccine consisting of a single or limited number ofpeptide specificities could theoretically be effective for protectingpopulations differing in HLA type (Loftus et al. 1995; Gulukota et al.1996; Sidney et al. 1996c).

The majority of information collected from examining divergent HLA-B15allotypes of sHLA molecules recombinantly produced according to themethodology of the present invention demonstrates that similar andoccasionally identical peptide ligands are presented by the differentB15 molecules so long as polymorphisms do not alter C-terminal anchoringpockets and while an N-proximal ligand residue can be subsequentlyanchored within the binding groove. Supporting data furthermoreindicates that these principles additionally extend beyond the HLA-B15allotypes described in specificity herein. Specifically, unpublishedresults by Ghosh and Wiley (noted in Bouvier and Wiley 1994) indicatethat an octamer has been observed to successfully bind a class Imolecule by its C terminus despite being shown through x-raycrystallography to not even reach the N-terminal pocket of the bindinggroove. In addition, a recently-described HIV-gag₁₉₇₋₂₀₅ CTL epitopepresented by murine class I K^(d) fails to show a motif-prescribed Tyrat P2 and instead associates stably through its conserved C terminus andan N-proximal preference for Gln at P3 (Mata et al. 1998). The rulesestablished here through examination of hundreds of natural ligands fromB*1501, B*1503, B*1508, B*1510, and B*1512 indicate that suchoccurrences may be more commonplace than exception, as both of theseexamples appear in agreement with the model in FIG. 27.

A step in developing therapies intended to elicit protective CTLsrequires the selection of pathogen- and tumor-specific peptide ligandsfor presentation by MHC class I and class II molecules.Binding/reconstitution assays provide information that is biased due totheir technical inconsistency and/or in vitro nature, while Edmansequencing of extracted class I peptide pools generates “motifs” thatindicate that the optimal peptides are nonameric ligands bearingconserved P2 and P9 anchors; motifs have frequently been used to providethe search parameters for selecting potentially immunogenic epitopesthat might be successfully presented by particular allotypes (Pamer etal. 1991; DiBrino et al. 1994; Kast et al. 1994; Davenport et al. 1995;Walden 1996; Holland et al. 1997; Zhang et al. 1997; Yoon et al. 1998;Chang et al. 1999). Therefore, to test the hypothesis that naturalpresentation overlaps exist despite the presence of variouspolymorphisms within the class I binding groove and thus determine howwell pooled motifs actually represent their endogenously-derivedconstituents, ligands were purified from different sHLA moleculesproduced in hollow-fiber bioreactors, mapped by RP-HPLC and NanoES-MS,and sequenced by NanoES-MS/MS, all according to the methodology of thepresent invention.

Production of sHLA provides an efficient means of extracting largequantities of endogenous peptide ligands for the subsequent analyses,and comparative ion mapping of peptides extracted from distinct class Iallotypes is a reliable method for detecting potential ligand overlaps.NanoES-MS/MS analysis then allows for sequence characterization toidentify the overlap status of individual ion matches. The strategydeveloped to address overlap identification is additionally pertinentbeyond the uses described herein. For example, similar mapping studieswould be performed, with the primary intent instead of characterizingdifferences between maps, such as between pathogenically infected versusuninfected cell lines; the data obtained could contribute to identifyingoptimal vaccine epitopes. This concept is currently in the early stagesof pursuit by several independent research teams (Veronese et al. 1996;van der Heeft et al. 1998). Successfully locating differences in asimilar manner between B*1501 and B*1512 ion maps, as discussed herein,effectively supports this application.

FIG. 28 summarizes the α-chain substitutions and motif-derived P2 and P9anchors for the HLA-B15 allotypes examined here, as well as additionalallotypes indicated earlier in FIG. 5 that appear to serve asevolutionary intermediates between or extensions from B*1503, B*1510,B*1512, and B*1518. B*4601 is included since it differs from B*1501 by asingle mutagenic event and overlaps with it were among peptidescharacterized in this study (Table 4). In section A, the allotypesB*1501, B*1503, B*1508, B*1512, and B*4601 have been shown to bindvariously overlapping ligands; based upon the structurally-predictedmotif anchors of the remaining molecules in this section includingB*1519, B*1529, B*1539, and B*1547, it is suspected that ion mapping andcharacterization would reveal further overlaps according to the modelshown in FIG. 27. Likewise, in section C the allotypes B*1509 and B*1510have demonstrated overlapping ligands and will probably share some withB*1537.

Systematically mapping and characterizing 449 ligands from the relatedmolecules B*1501, B*1503, B*1508, B*1510, and B*1512 demonstratesoverall that the peptides bound by these allotypes: (i) vary in lengthfrom 7 to 12 residues; and (ii) are more conserved at their C terminithat at their N-proximal positions. Flexibility at P2 in particularappears to arise at least in part from the combined effects of distinctsteric and charge biases imposed respectively by α-helical and β-sheetstructural residues throughout α₁ and α₂ of the various HLA-B15molecules, while it is postulated that C-terminal preferences areinfluenced by tapasin-moderated loading selection within the ER.

Although not predictable from the pooled Edman motifs, the comparativepeptide mapping strategy succeeded in identifying endogenously processedligands which bind variously across the allotypes B*1501, B*1503,B*1508, and B*1512, but not B*1510. Overlapping peptide ligands appearedto favorably bind the first four B15 molecules since these allotypesshare identical C-terminal anchoring pockets, whereas B*1510 diverges inthis region. Endogenous peptide loading into the HLA-B15 allotypestherefore requires that a conserved C terminus be firmly anchored in theappropriate specificity pocket while N-proximal residues act moreflexibly in terms of both location and sequence specificity to anchorthe ligand into this binding groove region. Subsequently, the choice ofallele-specific and/or overlapping peptide epitopes for CTL recognitionmay thus be contingent upon performing queries strongly based uponconserved C-terminal anchors.

Another embodiment of the present invention, as previously describedherein-above, is the use of genomic DNA (gDNA) as the starting materialfor the production of the sHLA molecules described hereinbefore.

This alternative method of the present invention begins by obtaininggenomic DNA which encodes the desired MHC class I or class II molecule.Alleles at the locus which encode the desired MHC molecule are PCRamplified in a locus specific manner. These locus specific PCR productsmay include the entire coding region of the MHC molecule or a portionthereof. In some cases a nested or hemi-nested PCR is applied to producea truncated form of the class I or class II gene so that it will besecreted rather than anchored to the cell surface. In other cases thePCR will directly truncate the MHC molecule.

Locus specific PCR products are cloned into a mammalian expressionvector and screened with a variety of methods to identify a cloneencoding the desired MHC molecule. The cloned MHC molecules are DNAsequenced to insure fidelity of the PCR. Faithful truncated (i.e., sHLA)clones of the desired MHC molecule are then transfected into a mammaliancell line. When such cell line is transfected with a vector encoding arecombinant class I molecule, such cell line may either lack endogenousclass I expression or express endogenous class I. It is important tonote that cells expressing endogenous class I may spontaneously releaseMHC into solution upon natural cell death. In cases where this smallamount of spontaneously released MHC is a concern, the transfected classI MHC molecule can be “tagged” such that it can be specifically purifiedaway from spontaneously released endogenous class I molecules in cellsthat express class I molecules. For example, a DNA fragment encoding aHis tail which will be attached to the protein may be added by the PCRreaction or may be encoded by the vector into which the gDNA fragment iscloned, and such His tail will further aid in purification of the classI molecules away from endogenous class I molecules. Tags beside ahistidine tail have also been demonstrated to work and are logical tothose skilled in the art of tagging proteins for downstreampurification.

Cloned genomic DNA fragments contain both exons and introns as well asother non-translated regions at the 5′ and 3′ termini of the gene.Following transfection into a cell line which transcribes the genomicDNA (gDNA) into RNA, cloned genomic DNA results in a protein productthereby removing introns and splicing the RNA to form messenger RNA(mRNA), which is then translated into an MHC protein. Transfection ofMHC molecules encoded by gDNA therefore facilitates reisolation of thegDNA, mRNA/cDNA, and protein.

Production of MHC molecules in non-mammalian cell lines such as insectand bacterial cells requires cDNA clones, as these lower cell types donot have the ability to splice introns out of RNA transcribed from agDNA clone. In these instances the mammalian gDNA transfectants of thepresent invention provide a valuable source of RNA which can be reversetranscribed to form MHC cDNA. The cDNA can then be cloned, transferredinto cells, and then translated into protein. In addition to producingsecreted MHC, such gDNA transfectants therefore provide a ready sourceof mRNA, and therefore cDNA clones, which can then be transfected intonon-mammalian cells for production of MHC. Thus, the present inventionwhich starts with MHC genomic DNA clones allows for the production ofMHC in cells from various species.

A key advantage of starting from gDNA is that viable cells containingthe MHC molecule of interest are not needed. Since all individuals inthe population have a different MHC repertoire, one would need to searchmore than 500,000 individuals to find someone with the same MHCcomplement as a desired individual—this is observed when trying to finda match for bone marrow transplantation. Thus, if it is desired toproduce a particular MHC molecule for use in an experiment ordiagnostic, a person or cell expressing the MHC allele of interest wouldfirst need to be identified. Alternatively, in the method of the presentinvention, only a saliva sample, a hair root, an old freezer sample, orless than a milliliter (0.2 ml) of blood would be required to isolatethe gDNA. Then, starting from gDNA, the MHC molecule of interest couldbe obtained via a gDNA clone as described herein, and followingtransfection of such clone into mammalian cells, the desired proteincould be produced directly or in mammalian cells or from cDNA in severalspecies of cells using the methods of the present invention describedherein.

Current experiments to obtain an MHC allele for protein expressiontypically start from mRNA, which requires a fresh sample of mammaliancells that express the MHC molecule of interest. Working from gDNA doesnot require gene expression or a fresh biological sample. It is alsoimportant to note that RNA is inherently unstable and is not easilyobtained as is gDNA. Therefore, if production of a particular MHCmolecule starting from a cDNA clone is desired, a person or cell linethat is expressing the allele of interest must traditionally first beidentified in order to obtain RNA. Then a fresh sample of blood or cellsmust be obtained; experiments using the methodology of the presentinvention show that ≧5 milliliters of blood that is less than 3 days oldis required to obtain sufficient RNA for MHC cDNA synthesis. Thus, bystarting with gDNA, the breath of MHC molecules that can be readilyproduced is expanded. This is a key factor in a system as polymorphic asthe MHC system; hundreds of MHC molecules exist, and not all MHCmolecules are readily available from mRNA. This is especially true ofMHC molecules unique to isolated populations or of MHC molecules uniqueto ethnic minorities. Starting class I or class II protein expressionfrom the point of genomic DNA simplifies the isolation of the gene ofinterest and insures a more equitable means of producing MHC moleculesfor study; otherwise, one would be left to determine whose MHC moleculesare chosen and not chosen for study, as well as to determine whichethnic population from which fresh samples cannot be obtained should nothave their MHC molecules included in a diagnostic assay.

While cDNA may be substituted for genomic DNA as the starting material,production of cDNA for each of the desired HLA class I types willrequire hundreds of different, HLA typed, viable cell lines, eachexpressing a different HLA class I type. Alternatively, fresh samplesare required from individuals with the various desired MHC types. Theuse of genomic DNA as the starting material allows for the production ofclones for many HLA molecules from a single genomic DNA sequence, as theamplification process can be manipulated to mimic recombinatorial andgene conversion events. Several mutagenesis strategies exist whereby agiven class I gDNA clone could be modified at either the level of gDNAor at the cDNA resulting from this gDNA clone. The process of thepresent invention does not require viable cells, and therefore thedegradation which plagues RNA is not a problem. Thus, from a given gDNAclone, any number of gDNA and cDNA MHC molecules can be produced.

Three useful products can be obtained from the mammalian cell lineexpressing HLA class I molecules from such a genomic DNA construct. Thefirst product is the soluble class I MHC protein, which may be purifiedand utilized in various experimental strategies, including but notlimited to epitope testing. Epitope testing is a method for determininghow well discovered or putative peptide epitopes bind individual,specific class I or class II MHC proteins. Epitope testing with secretedindividual MHC molecules has several advantages over the prior art,which utilized MHC from cells expressing multiple membrane-bound MHCs.While the prior art method could distinguish if a cell or cell lysatewould recognize an epitope, such method was unable to directlydistinguish in which specific MHC molecule the peptide epitope wasbound. Lengthy purification processes might be used to try and obtain asingle MHC molecule, but doing so limits the quantity and usefulness ofthe protein obtained. The novelty of the current approach is thatindividual MHC specificities can be utilized in sufficient quantitythrough the use of recombinant, soluble MHC proteins. Because MHCmolecules participate in numerous immune responses, studies of vaccines,transplantation, immune tolerance, and autoimmunity can all benefit fromindividual MHC molecules provided in sufficient quantity.

A second important product obtained from mammalian cells secretingindividual MHC molecules is the peptide cargo carried by MHC molecules.Class I and class II MHC molecules are really a trimolecular complexconsisting of an alpha chain, a beta chain, and the alpha/beta chain'speptide cargo to be reviewed by immune effector cells. Since it is thepeptide cargo, and not the MHC alpha and beta chains, which marks a cellas infected, tumorigenic, or diseased, there is a great need tocharacterize the peptides bound by particular MHC molecules. Forexample, characterization of such peptides will greatly aid indetermining how the peptides presented by a person with MHC-associateddiabetes differ from the peptides presented by the MHC moleculesassociated with resistance to diabetes. As stated above, having asufficient supply of an individual MHC molecule, and therefore that MHCmolecules bound peptides, provides a means for studying such diseases.Because the method of the present invention provides quantities of MHCprotein previously unobtainable, unparalleled studies of MHC moleculesand their important peptide cargo can now be facilitated.

The methodology for producing sHLA from gDNA, while similar to themethodology for producing sHLA from cDNA, is different and as suchrequires different and/or unique steps and/or processes for itscompletion. One exemplary detailed production methodology for use gDNAas the starting material for the production of MHC class I or IImolecules is described herein below.

Genomic DNA Extraction

Greater than or equal to 200 ul of sample either blood, plasma, serum,buffy coat, body fluid or up to 5×10⁶ lymphocytes in 200 ul phosphatebuffered saline was used to extract genomic DNA using the QlAampr DNABlood Mini Kit blood and body fluid spin protocol. Genomic DNA qualityand quantity was assessed using optical density readings at 260 nm and280 nm

PCR Strategy

Primers have been designed for HLA-A, -B and -C loci in order to producea truncated amplicon of the human class I MHC using a two-stage PCRstrategy. The first stage PCR uses a primer set that amplify from the 5′Untranslated region to Intron 4. This amplicon is used as a template forthe second PCR which results in a truncated version of the MHC Class Igene by utilizing a 3′ primer that sits down in exon 4 at codon 298(including the leader peptide), the 5′ primer remains the same as the1^(st) PCR. An overview of this PCR strategy can be seen in FIG. 29. Theprimers for each locus are listed in Table 17. Different HLA-B locusalleles require primers with different restriction cut sites dependingon the nucleotide sequence of the allele. Hence there are two 5′ and two3′ truncating primers for the HLA-B locus.

1. Primary PCR Materials

An Eppendorf Gradient Mastercycler is used for all PCR. H₂O: Dionizedultra filtered water (DIUF) Fisher Scientific, W2-4,41. PCR nt mix (10mM each deoxyribonucleoside triphosphate [dNTP]), Boehringer Manheim,#1814, 362. 10× Pfx Amplification buffer, pH 9.0, GibcoBRLR, part#52806, formulation is proprietary information. 50 mM MgSO₄, GibcoBRLR,part #52044. Platinumâ Pfx DNA Polymerase (B Locus only), GibcoBRLR,11708-013. Pfu DNA Polymerase (A and C Locus), Promega, M7741. Pfu DNAPolymerase 10× reaction Buffer with MgSO₄, 200 mM Tris-HCL, pH 8.8, 100mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1 mg/ml nuclease free BSA, 1%TritonrX-100. gDNA Template. Amplification primers (in ng/ul):

a. A locus: 5′ sense PP5UTA (300); 3′antisense PPI4A (300)b. B locus (Not B*39's): sense PP5UTB (300); antisense PPI4B (300)c. B locus (B*39's): sense 5UTB39 (300); antisense PPI4B (300)d. C Locus: sense 5PKCE (300); antisense PPI4C (300).

B. Secondary PCR (Also Used for Colony PCR)

H₂O: Dionized ultra filtered water (DIUF) Fisher Scientific, W2-4,41.PCR nt mix (10 mM each deoxyribonucleoside triphosphate [dNTP]),Boehringer Manheim, #1814, 362. Pfu DNA Polymerase, Promega, M7741. PfuDNA Polymerase 10× reaction Buffer with MgSO₄, 200 mM Tris-HCL, pH 8.8,100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1 mg/ml nuclease free BSA, 1%TritonrX-100. Template 1:100 dilution of the primary PCR product.Amplification primers (in ng/ul):

a. A-locus: 5′ sense PP5UTA (300), 3′ antisense PP3PEI (300)b. B-locus: sense PP5UTB (300), antisense PP3PEI (300)c. B-locus: sense PP5UT (300), antisense PP3PEIH (300)d. B-locus B39's: sense 5UTB39 (300), antisense PP3PEIH (300)e. C-locus: sense 5PKCE (300), antisense PP3PEI (300)f. C-locus Cw*7's: sense 5PKCE (300), antisense 3PEIHC7 (300)

C. Gel Purification of PCR Products and Vectors

Dark Reader Transilluminator Model DR-45M, Clare Chemical Research. SYBRGreen, Molecular Probes Inc. Quantum Prep Freeze 'N Squeeze DNA GelExtraction Spin Columns, Bio-Rad Laboratories, 732-6165.

D. Restriction Digests, Ligation and Transformation

Restriction enzymes from New England Biolabs: EcoR I #R0101S; Hind III#R0104S; Xba I #R0145S. T4 DNA Ligase, New England Biolabs, #M0202S.pcDNA3.1(−), Invitrogen Corporation, V795-20. 10× Buffers from NewEngland Biolabs. EcoR I buffer, 500 mM NaCl, 1000 mM Tris-HCL, 10 mMMgCL₂, 0.25% Triton-X 100, pH 7.5. T4 DNA ligase buffer, 500 mMTris-HCL, 100 mM MgCL₂, 100 mM DTT, 10 mM ATP, 250 ug/ml BSA, pH 7.5.NEB buffer 2, 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl₂, 10 mM DDT, pH7.9. 100×BSA, New England Biolabs. Z-Competent E. coli TransformationBuffer Set, Zymo Research, T3002. E. coli strain JM109. LB Plates with100 ug/ml ampicillin. LB media with 100 ug/ml ampicillin

E. Plasmid Extraction

Wizard Plus SV minipreps, Promega, #A1460

3. Sequencing of Clones

Thermo Sequenase Primer Cycle Sequencing Kit, Amersham PharmaciaBiotech, 25-2538-01. CY5 labeled primers (Table 18). Alfexpressautomated DNA sequencer, Amersham Pharmacia Biotech.

4. Gel Casting

PagePlus 40% concentrate, Amresco, E562, 500 ml. Urea, AmershamPharmacia Biotech, 17-0889-01,500 g. N′N′N′N′-tetramethylethyleneiamine(TEMED), APB. Ammonium persulphate (10% solution), APB. Boric acid, APB.EDTA-disodium salt, APB. Tris, APB. Bind-Saline, APB. Isopropanol,Sigma. Glacial Acetic Acid, Fisher Biotech. DIUF water, FisherScientific. Ethanol 200-proof.

5. Plasmid Preparation for Electroporation

Qiagen Plasmid Midi kit, Qiagen Inc., 12143.

A. Electroporation

Biorad Gene Pulser with capacitance extender, Bio-Rad Laboratories. GenePulser Cuvette, Bio-Rad Laboratories. Cytomix: 120 mM KCl, 0.15 mMCaCl₂, 10 mMK₂HPO₄/KH₂PO₄, pH 7.6, 25 mM Hepes, pH 7.6, 2 mM EGTA, pH7.6, 5 mM MgCl₂, pH 7.6 with KOH. RPMI 1640+20% Foetal CalfSerum+Pen/strep. Haemacytometer. Light Microscope. CO₂ 37° Incubator.Cells in log phase.

Methods used for the production of soluble human HLA Class I and IIproteins in mammalian cells from gDNA.

1. Primary PCR

A. A-Locus and C-Locus

10x Pfu buffer 5 ul 5′ Primer (300 ng/ul) 1 ul 3′ Primer (300 ng/ul) 1ul dNTP's (10 mM each) 1 ul gDNA (>>50 ng/ul) 10 ul  DIUF H₂0 31.4 ul  Pfu DNA Polymerase 0.6 ul   96° C. 2 min. 95° C. 1 min x35 58° C. 1 minx35 73° C. 5 min x35 73° C. 10 min

B. B-locus

10x Pfx buffer 5 ul 5′ Primer (300 ng/ul) 1 ul 3′ Primer (300 ng/ul) 1ul dNTP's (10 mM each) 1.5 ul   MgSO₄ (50 mM) 1 ul gDNA (100 ng/ul) 1 ulDIUF H₂O 40 ul  Pfx DNA Polymerase 0.5 ul   94° C. 2 min. 94° C. 1 minx35 60° C. 1 min x35 68° C. 3.5 min x35 68° C. 5 min

2. Gel Purification of PCR (all PCR and Plasmids are Gel Purified)

Mix primary PCR with 5 ul of 10×SYBR green and incubate at roomtemperature for 15 minutes then load on a 2% agarose gel. Visualize onthe Dark Reader and purify using the Quantum Prep Freeze and Squeezeextraction kit according to the manufacturers instructions.

3. Secondary PCR of A, B and C Loci

10x Pfu buffer   5 ul 5′ Primer (300 ng/ul) 1.0 ul 3′ Primer (300 ng/ul)1.0 ul dNTP's (10 mM each)   1 ul 1:100 1° PCR  10 ul DIUF H₂0 31.4 ul Pfu DNA Polymerase 0.6 ul 96° C. 2 min. 95° C. 1 min x35 56° C. 1 minx35 73° C. 4 min x35 73° C. 7 min

4. Restriction Digests

2° PCR (gel purified) 30 ul Restriction enzyme 1 X ul Restriction enzyme2 X ul 10x buffer 5 ul 100x BSA 0.5 ul DIUF H₂O 10.5 ul

The cut sites incorporated into the PCR primers for each individual PCRwill determine the enzymes used. The expression vector pcDNA3.1(−) willbe cut in a similar manner.

5. Ligation

pcDNA3.1(—) cut with same enzymes as PCR x ng Cut PCR y ng 10x T4 DNAligase buffer 2 ul T4 DNA Ligase 1 ul DIUF H₂0 up to 20 ul

The ratio of vector to insert will vary between samples, a good startingpoint is a ratio of 1:6

6. Transformation

Transform JM109 using competent cells made using Z-competent E. coliTransformation Kit and Buffer Set.

7. Colony PCR and Restriction Digests

Either following the secondary PCR protocol or carrying out anotherrestriction digest can be used to screen the vector for the appropriateinsert.

8. Mini Preps of Colonies with Insert

Use the Wizard Plus SV minipreps and follow the manufacturersinstructions. Make glycerol stocks before beginning extraction protocol.

9. Sequencing of Positive Clones and Gel Casting

Using the Thermo Sequenase Primer Cycle Sequencing Kit

A, C, G or T mix 3 ul CY5 Primer 1 pm/ul 1 ul DNA template 100 ng/ul 5ul 96° C. 2 min 96° C. 30 sec x25 61° C. 30 sec x25

Add 6 ul formamide loading buffer and load 10 ul onto sequencing gelAnalyse sequence for good clones with no misincorporations.

10. Gel Casting

A. Prepare a 10×TBE stock solution for the sequencing gel mix:

500 mL Tris 60.5 g EDTA 1.85 g Boric Acid 25.5 g Fisher DIUF H₂O 440 mL

Filter using a 0.22 m or 0.45 m filter and store at 4° C. untilrequired.

11. Prepare a 10×TBE Stock Solution for the Running Buffer:

1 L Tris 121.0 g EDTA 3.7 g Boric Acid 51.0 g Ultra pure H₂O 880 mL

12. Prepare the Sequencing Gel Mix:

1 Gel, 6% PagePlus Urea 19.8 g PagePlus 40% conc. 7.95 mL 10X TBE 5.5 mLFisher DIUF H₂O 25.3 mL Filter using a 0.22 m or 0.45 m filter.

Initiate polymerization of the sequencing gel by adding 330 uL of afreshly made 10% APS solution and 33 uL of TEMED. Cast a 0.5 mmsequencing gel and allow it to polymerize for 5 hours.

13. Midi Preps

Prepare plasmid for electroporation using the Qiagen Plasmid Midi Kitaccording to the manufacturers instructions.

14. Electroporation

Electroporations are performed as described in “The Bw4 public epitopeof HLA-B molecules confers reactivity with natural killer cell clonesthat express NKb1, a putative HLA receptor. Gumperz, J. E., V. Litwin,J. H. Phillips, L. L. Lanier and P. Parham. J. Exp. Med. 181:1133-1144,1995,” which is herein expressly incorporated by reference in itsentirety.

15. Screening for Production of Soluble HLA

An ELISA is used to screen for the production of soluble HLA, see ELISAprotocol.

ELISA Protocol Solutions: 3 N H₂SO₄: For 500 ml: 200 ml H₂O 300 ml 5 NH₂SO₄ Store at room-temperature 10x PBS (pH 7.4): 26.8 mM KCl 14.7 mMKH₂PO₄ 1.37 M NaCl 81 mM Na₂HPO₄ For 1000 ml: 2 g KCl 2 g KH₂PO₄anhydrous 80 g NaCl 11.5 g Na₂HPO₄ Add H₂O Adjust pH to 7.4 Add up withH₂O to 1000 ml Filter Store at 4° C. TBS coating buffer (pH 8.5): 25 mMTris-HCl pH 8.5 150 mM NaCl For 1000 ml: 100 ml 10x TBS pH 8.5 900 mlH₂O Store at 4° C. 10% BSA For 250 ml: 25 g BSA Add H₂O Stir to dissolveAdd H₂O up to 225 ml Filter Store at 4° C. (Add 10x PBS prior to use)ELISA WASH 2.68 mM KCl 1.47 mM KH₂PO₄ 137 mM NaCl 8.1 mM Na₂HPO₄ 0.05%Tween-20 For 4000 ml: 400 ml 10x PBS 3598 ml H₂O 2 ml Tween-20 Store at4° C. OPD Substrate Solution for HRP: 0.05 M Na₂HPO₄ pH 5.0 0.025 MCitrate 0.4 mg/ml OPD 0.012% H₂O₂ For 10 ml: 10 ml 0.05 M Phosphate- pH5.0 citrate buffer 2 tablets OPD (2 mg ea) (Sigma; P-6787) 4 μl 30%Hydrogen (Sigma; H1009) peroxide (H₂O₂) Always prepare fresh Use within1 hour of preparation Add fresh 30% H₂O₂ immediately prior to use 0.05 MPhosphate-Citrate buffer: 0.05 M Na₂HPO₄ pH 5.0 0.025 M Citrate For 1000ml: 7.10 g Na₂HPO₄ anhydrous (Dibasic) 5.25 g Citric acid monohydrateAdd ddH₂O Adjust pH to 5.0, if necessary Add ddH₂O to a final volume of1000 ml Filter Store at room temperature

The HLA ELISA Procedure

For biochemical analysis, monomorphic monoclonal antibodies areparticularly useful for identification of HLA locus products and theirsubtypes W6/32 is one of the most common monoclonal antibodies (mAb)used to characterize human class I major histocompatibility complex(MHC) molecules. It is directed against monomorphic determinants onHLA-A, -B and -C heavy chains, which recognizes only mature complexedclass I molecules and recognizes a conformational epitope on the intactMHC molecule containing both beta2-microglobulin (b2m) and the heavychain (HC). W6/32 binds a compact epitope on the class I molecule thatincludes both residue 3 of beta2m and residue 121 of the heavy chain(Ladasky J J, Shum B P, Canavez F, Seuanez H N, Parham P. Residue 3 ofbeta2-microglobulin affects binding of class I MHC molecules by theW6/32 antibody. Immunogenetics 1999 April; 49(4):312-20.). The constantportion of the molecule W6/32 binds to is recognized by CTLs and thuscan inhibit cytotoxicity. The reactivity of W6/32 is sensitive to theamino terminus of human beta2-microglobulin (Shields M J, Ribaudo R K.Mapping of the monoclonal antibody W6/32: sensitivity to the aminoterminus of beta2-microglobulin. Tissue Antigens 1998 May;51(5):567-70). W6/32 is available biotinylated (Serotec MCA81B) offeringadditional variations in ELISA procedures.

Anti-human b2m (HRP) (DAKO P0174) recognizes denatured as well ascomplexed b2m. Although in principle anti-b2m reagents could be used forthe purpose of identification of HLA molecules, they are less suitablewhen association of heavy chain and b2m is weak. The patterns of class Imolecules precipitated with W6/32 and anti-b2m are usuallyindistinguishable [Vasilov, 1983 #10].

Rabbit anti-b2-microglobulin dissociates b2-microglobulin from heavychain as a consequence of binding (Rogers, M. J., Appella, E., Pierotti,M. A., Invernizzi, G., and Parmiani, G. (1979) Proc Natl. Acad. Sci.U.S.A. 76, 1415-1419). It also has been reported that rabbit anti-humanb2-microglobulin dissociates b2-microglobulin from HLA heavy chains uponbinding (Nakamuro, K., Tanigaki, N., and Pressman, D. (1977) Immunology32, 139-146.). This anti-human b2m antibody is also availableunconjugated (DAKO A0072).

The W6/32-HLA Sandwich ELISA

Sandwich assays can be used to study a number of aspects of proteincomplexes. If antibodies are available to different components of aheteropolymer, a two-antibody assay can be designed to test for thepresence of the complex. Using a variation of these assays, monoclonalantibodies can be used to test whether a given antigen is multimeric.

The W6/32-anti-b2m antibody sandwich assay is one of the best techniquesfor determining the presence and quantity of sHLA. Two antibody sandwichassays are quick and accurate, and if a source of pure antigen isavailable, the assay can be used to determine the absolute amounts ofantigen in unknown samples. The assay requires two antibodies that bindto non-overlapping epitopes on the antigen. This assay is particularlyuseful to study a number of aspects of protein complexes.

To detect the antigen (sHLA), the wells of microtiter plates are coatedwith the specific (capture) antibody W6/32 followed by the incubationwith test solutions containing antigen. Unbound antigen is washed outand a different antigen-specific antibody (anti-b2m) conjugated to HRPis added, followed by another incubation. Unbound conjugate is washedout and substrate is added. After another incubation, the degree ofsubstrate hydrolysis is measured. The amount of substrate hydrolyzed isproportional to the amount of antigen in the test solution.

The major advantages of this technique are that the antigen does notneed to be purified prior to use and that the assays are very specific.The sensitivity of the assay depends on 4 factors:

-   -   (1) The number of capture antibody    -   (2) The avidity of the capture antibody for the antigen    -   (3) The avidity of the second antibody for the antigen    -   (4) The specific activity of the labeled second antibody        Use an ELISA protocol template and label a clear 96-well        polystyrene assay plate. Polystyrene is normally used as a        microtiter plate. (Because it is not translucent, enzyme assays        that will be quantitated by a plate reader should be performed        in polystyrene and not PVC plates).

Company Plate Specificity Cat# Nunc Maxisorp standard/untreated 441653StarWell Modules Framed 8-well strips

Coating of the W6/32 should be performed in Tris buffered saline (TBS);pH 8.5. Prepare a coating solution of 8.0 μg/ml of specific W6/32antibody in TBS (pH 8.5). Although this is well above the capacity of amicrotiter plate, the binding will occur more rapidly. Higherconcentrations will speed the binding of antigen to the polystyrene butthe capacity of the plastic is only about 100 ng/well (300 ng/cm²), sothe extra protein will not bind. If using W6/32 of unknown compositionor concentration, first titrate the amount of standard antibody solutionneeded to coat the plate versus a fixed, high concentration of labeledantigen. Plot the values and select the lowest level that will yield astrong signal. Do not include sodium azide in any solutions whenhorseradish peroxidase is used for detection.

Immediately coat the microtiter plate with 100 μl per well using amultichannel pipet. Standard polystyrene will bind antibodies orantigens when the proteins are simply incubated with the plastic. Thebonds that hold the proteins are non-covalent, but the exact types ofinteractions are not known. Shake the plate to ensure that the antigensolution is evenly distributed over the bottom of each well. Seal theplate with plate sealers (sealplate adhesive sealing film, nonsterile,100 per unit; Phenix (1-800 767-0665); LMT-Seal-EX) or sealing tape toNunc-Immuno™ Modules (#236366). Incubate at 4° C. overnight. Avoiddetergents and extraneous proteins. Next day, remove the contents of thewell by flicking the liquid into the sink or a suitable waste container.Remove last traces of solution by inverting the plate and blotting itagainst clean paper toweling. Complete removal of liquid at each step isessential for good performance. Wash the plate 10 times with Wash Buffer(PBS containing 0.05% Tween-20) using a multi-channel ELISA washer.

After the last wash, remove any remaining Wash Buffer by inverting theplate and blotting it against clean paper toweling. After the W6/32 isbound, the remaining sites on the plate must be saturated by incubatingwith blocking buffer made of 3% BSA in PBS. Fill the wells with 200 μlblocking buffer. Cover the plates with an adhesive strip and incubateovernight at 4° C. Alternatively, incubate for at least 2 hours at roomtemperature which is, however, not the standard procedure. Blockedplates may be stored for at least 5 days at 4° C. Good pipettingpractice is most important to produce reliable quantitative results. Thetips are just as important a part of the system as the pipette itself.If they are of inferior quality or do not fit exactly, even the bestpipette cannot produce satisfactory results.

When maximum levels of accuracy are stipulated, prewetting should beused at all times. To do this, the required set volume is first drawn inone or two times using the same tip and then returned. Prewetting isabsolutely necessary on the more difficult liquids such as 3% BSA. Donot prewet, if your intention is to mix your pipetted sample thoroughlywith an already present solution. However, prewet only for volumesgreater than 10 μl. In the case of pipettes for volumes less than 10 μlthe residual liquid film is as a rule taken into account when designingand adjusting the instrument. The tips must be changed between eachindividual sample. With volumes <10 μl special attention must also bepaid to drawing in the liquid slowly, otherwise the sample will besignificantly warmed up by the frictional heat generated. Then slowlywithdraw the tip from the liquid, if necessary wiping off any dropsclinging to the outside. To dispense the set volume hold the tip at aslight angle, press it down uniformly as far as the first stop.

In order to reduce the effects of surface tension, the tip should be incontact with the side of the container when the liquid is dispensed.After liquid has been discharged with the metering stroke, a short pauseis made to enable the liquid running down the inside of the tip tocollect at its lower end. Then press it down swiftly to the second stop,in order to blow out the tip with the extended stroke with which theresidual liquid can be blown out. In cases that are not problematic(e.g., aqueous solutions) this brings about a rapid and virtuallycomplete discharge of the set volume. In more difficult cases, a slowerdischarge and a longer pause before actuating the extended stroke canhelp. To determine the absolute amount of antigen (sHLA), sample valuesare compared with those obtained using known amounts of pure unlabeledantigen in a standard curve.

For accurate quantization, all samples have to be run in triplicate, andthe standard antigen-dilution series should be included on each plate.Pipetting should be preformed without delay to minimize differences intime of incubation between samples. All dilutions should be done inblocking buffer. Thus, prepare a standard antigen-dilution series bysuccessive dilutions of the homologous antigen stock in 3% BSA in PBSblocking buffer. In order to measure the amount of antigen in a testsample, the standard antigen-dilution series needs to span most of thedynamic range of binding. This range spans from 5 to 100 ng sHLA/ml.

A stock solution of 1 μg/ml should be prepared, allocated in volumes of300 μl and stored at 4° C. Prepare a 50 ml batch of standard at thetime. (New batches need to be compared to the old batch before used inquantization). Use a tube of the standard stock solution to preparesuccessive dilutions according to the scheme below. While standardcurves are necessary to accurately measure the amount of antigen in testsamples, they are unnecessary for qualitative “yes/no” answers.

For accurate quantization, the test solutions containing sHLA should beassayed over a number of at least 4 dilutions to assure to be within therange of the standard curve. Prepare serial dilutions of each antigentest solution in blocking buffer (3% BSA in PBS). Standard dilutions forpurified, crude or flow through samples are given below:

After mixing, prepare all dilutions in disposable U-bottom 96 wellmicrotiter plates before adding them to the W6/32-coated plates with amulti pipette. Add 150 μl in each well. To further proceed, remove anyremaining blocking buffer and wash the plate as described above. Theplates are now ready for sample addition. Add 100 μl of the sHLAcontaining test solutions and the standard antigen dilutions to theantibody-coated wells. Cover the plates with an adhesive strip andincubate for exactly 1 hour at room temperature. After incubation,remove the unbound antigen by washing the plate 10× with Wash Buffer(PBS containing 0.05% Tween-20) as described.

Prepare the appropriate developing reagent to detect sHLA. Use thesecond specific antibody, anti-human b2m-HRP (DAKO P0174/0.4 mg/ml)conjugated to Horseradish Peroxidase (HRP). Dilute the anti-humanb2m-HRP in a ratio of 1:1'000 in 3% BSA in PBS. (Do not include sodiumazide in solutions when horseradish peroxidase is used for detection).

No. of Total 3% BSA plates Antibody anti-b2m-HRP in PBS Mix: 1 10 ml 10μl 10 ml 2 20 ml 20 μl 20 ml 3 30 ml 30 μl 30 ml 4 40 ml 40 μl 40 ml 550 ml 50 μl 50 ml

Add 100 μl of the secondary antibody dilution to each well. Alldilutions should be done in blocking buffer. Cover with a new adhesivestrip and incubate for 20 minutes at room temperature. Prepare theappropriate amount of substrate prior to the wash step. Bring thesubstrate to room temperature. OPD (o-Phenylenediamine) is a peroxidasesubstrate suitable for use in ELISA procedures. The substrate produces asoluble end product that is yellow in color. The OPD reaction is stoppedwith 3 N H₂SO₄, producing an orange-brown product and read at 492 nm.Prepare OPD fresh from tablets (Sigma, P6787; 2 mg/tablet). The solidtablets are convenient to use when small quantities of the substrate arerequired.

After second antibody incubation, remove the unbound secondary reagentby washing the plate 10× with Wash Buffer (PBS containing 0.05%Tween-20). After the final wash, add 100 μl of the OPD substratesolution to each well and allow to develop at room temperature for 10minutes. Reagents of the developing system are light-sensitive, thus,avoid placing the plate in direct light. Prepare the 3 N H₂SO₄ stopsolution. After 10 minutes, add 100 μl of stop solution per 100 μl ofreaction mixture to each well. Gently tap the plate to ensure thoroughmixing. Read the ELISA plate at a wavelength of 490 nm within a timeperiod of 15 minutes after stopping the reaction.

The background should be around 0.1. If the background is higher, thesubstrate may have been contaminated with a peroxidase. If the substratebackground is low and the background in your assay is high, this may bedue to insufficient blocking. Finally analyze your readings. Prepare astandard curve constructed from the data produced by serial dilutions ofthe standard antigen. To determine the absolute amount of antigen,compare these values with those obtained from the standard curve.

After running the ELISA on various transfectants it is possible todetermine which ones will produce the highest levels of soluble HLAmolecules. These “good producers” are grown to the point where a cellpharm can be seeded. The harvest from the cell pharm is then used toextract the soluble HLA using the following purification protocol.

To start the chromatography procedure, prepare the ÄKTA™ prime system.The system can be used immediately but the spectrophotometers fullability will not be obtained until after 1 hour of lamp warm-up. Toprepare the system for a run, check that the buffer inlet tubings areimmersed in the correct buffer vessels and the waste tubings are putinto a waste bottle. Only use degassed and filtered liquids to make surethat the liquid remains free from air bubbles. Degass by applying avacuum to the solution. Prepare and hook up the buffers necessary for asHLA purification:

Buffer Valve (A) 1. PBS, pH 7.4 (Wash buffer) 2. 20% Ethanol/70% Ethanol(Cleaning solutions) 3. 0.1 N NaO (MOK elution buffer) 4. 50 mMDiethylamine (MOK elution buffer) (DEA), pH 11.3 5. Protein sample (Theline is stored in PBS/ 0.05% Na Azide, pH 7.4) 6. 0.2 N Acetic acid, pH~2.7 (Cleaning & MOK solution) 7. 0.1 M Citric acid, pH 3.0 (Protein Aelution buffer) 8. 0.1 M Glycine, pH 11.0 (sHLA elution buffer)

It is important to purge the lines after a new hook-up with about 50 mlof liquid to get the air out of the system. Purging can be done manuallythrough the inlets of the buffer valve (A1-A8), while carefullyimmersing the tubing in the respective liquid. To remove any trapped airbubbles in the flow path, purge the pump in the order PBS/20%ethanol/PBS/final buffer solution. Next, prepare the recorder to monitorthe purification. Autozero the built-in UV spectrophotometer with PBS asreference. Equilibrate all material to the temperature at which thechromatography will be performed. For large scale purifications, attachthe column entrance/exit to the system.

Equilibrate the column by passing 10 bed-volumes of PBS over the matrix.Before starting any column purification, the protein concentration inthe sample solution should be determined using a quantitative ELISAprocedure. The sample volume loaded will depend on the size and loadingcapacity of the column and the concentration of the sample. Calculatethe volume of the sample solution maximally saturating the columnaccording to the columns capacity to bind the antigen.

(A) Antigen concentration: mg/ml antigen (B) Antigen binding capacity:mg antigen/ml gel (C) Matrix volume: ml gel (D) Maximal amount ofantigen: (B * C) mg (E) Sample volume: (D/A) ml

Since the binding capacity of the column will realistically not bereached, a much lower volume of sample solution should be chosen. Avalue between 40 to 50% of the calculated volume is more accurate whichalso will not result in the waste of lots of unbound antibody within theflow-through. Prepare the antibody sample solution for purification.Spin crude harvest at 5'000 rpm for 25 minutes (JA10 rotor) to removelipid and cell debris. The antigen solution must be free of particulatematter. Pour the supernatant into a suitable container. Prevent airbubble formation.

Name of the crude harvest: Volume used: ml Amount of sample: mg

The simplest method to bind the antigen to the antibody/Sepharose 4Bmatrix is to apply the sample through the system pump and pass theprotein solution down the column. Set appropriate parameters to recordthe loading conditions on the recorder.

Chart Speed Conductivity Optical Density Load 0.1 mm/min 0.5 V 1.0 V

Save a 1 ml probe from the starting material (LOAD) before thepurification procedure for analysis purposes. Set the buffer valve toposition 5 and the injection valve to position LOAD. Make sure the inlettubings are purged with sample buffer without any air-bubbles present.To have a purged sample line, disconnect shortly the column beforeloading and circulate the sample within the system with higher flowrate. Pass the solution slowly through the column with a flow rate ofapproximately 1.0 ml/min or lower to give the protein time to bind moreefficiently. Higher flow rates will decrease efficiency. A disruption inflow may cause a rapid rise in back-pressure. If this occurs,immediately shut off the pump and check the gel bed for compression.

Collect the flow-through in an appropriate container. Keep until you aresure all material has bound to the column and negligible amounts are inthe flow through. Take a sample at the end of the run (Ft) which shouldbe analyzed. Wash the column with PBS at 10 ml/min until UV absorbanceat 280 nm is zero. For a large column use 2000-3000 ml wash buffer(PBS). Save the wash in a container until after the purification.

Chart Speed Conductivity Optical Density Wash 0.5 mm/min 0.5 V 1.0 V

Prepare borosilicate collection tubes by adding 1.2 ml of 1 M Tris-HCl,pH 7.0 per 4.8 ml of fraction to be collected (1:4). Neutralization is asafety measure to preserve the activity of the eluted molecule. MHCclass I (sHLA) molecules are best eluted from a W6/32 column by 0.1 Mglycine, pH 11.0. Absorbance is used for generating a protein elutionprofile.

Chart Speed Conductivity Optical Density Elution 0.5 mm/sec 0.2 V 0.1 V

Place the collector arm over the first collection tube. Elute 4.8 ml perfraction at 10 ml/min. Immediately afterwards, mix each tube gently tobring the pH back to neutral. As with all protein solutions, avoidbubbling or frothing as this denatures the proteins. If a very lowamount of protein is expected, change the conductivity on the recorderto a lower value. Identify the antigen-containing fractions byabsorbance at 280 nm on the chart and combine them duringup-concentration. Up-concentrate immediately and buffer exchange intoPBS using MACROSEP™ centrifugal concentrators (Pall Filtron;Northborough, Mass.; MACROSEP 10K; OD010C37). Keep the protein on ice atall times and centrifuge at 4° C. After the buffer exchange, prepare thesample for storage at 4° C. Filter the pure samples through a 0.2μfilter and aliquot directly into sterile, screw cap tubes. Labelappropriately.

Determine the absorbance at 280 nm as well as the protein concentrationwith the Micro BCA kit. Activity can be determined with a regular ELISAprocedure. The purity of the eluted sHLA can be assessed by SDS-PAGE,Western blotting or performing a Superdex column analysis. After theelution, quickly re-equilibrate the column with PBS to avoiddenaturation of the W6/32 antibody linked to it. For analytical work inwhich more than one allele will be purified on the same column, extremecare must be taken. To be able to re-use the column, start a maintenanceprocedure after the re-equilibration. Cleaning-in-place is a procedure,which removes contaminants such as lipids, precipitates or denaturedproteins that may remain in the column after regeneration. Suchcontaminations are especially likely when working with crude materials.The procedure helps to maintain the capacity, flow properties andgeneral performance.

Mock elute the column using buffers with alternating pH. Start runningover 10 gel volumes of 0.2 N acetic acid followed by 10 gel volumes of50 mM diethylamine, pH 11.3 at a speed of 10 ml/min. Repeat three timesand always equilibrate with 10 gel volumes PBS between buffer changes.

Chart Speed Conductivity Optical Density Mok-elution 1.0 mm/min 0.2 V0.1 V

After Mok-elution, store the column at 4° C. in PBS/0.05% Na Azide.Sanitization inactivates microbial contaminants in the packed column andrelated equipment. One generally recommended procedure is to washalternately with high and low pH buffers as performed in the couplingreaction. For sanitization, disassemble the column and wash the matrixalternately with low pH wash buffer (0.1 M sodium acetate containing 0.5M NaCl, pH 4.0) and high pH wash buffer (0.1 M Tris-HCl containing 0.5 MNaCl, pH 8.0) for 3 times followed by re-equilibration with PBS.Re-assemble the cleaned and sterilized column and store it at 4° C. inPBS containing 0.05% sodium azide.

After the column is removed, the ÄKTA™ prime system has to be cleanedcarefully. Start with the cleaning of line 5, where the sample washooked up. Rinse the system pump and include the fraction collectorline. First clean the inlet tubings, by manually running the system pumpand flushing with 0.2 N acetic acid at 30 ml/min followed by 0.1 N NaOH.Always equilibrate with PBS. Don't forget to add a line between theinjection valve and the UV detector as a bridge, as replacement of thecolumn. Finally, rinse with 20% ethanol. If the column was sanitizedbecause of bacterial contamination, rinse with 70% ethanol.

In order to test whether or not the sHLA molecules produced wereconformational, an assay was developed using a variety of differentantibodies:

1. w6/32, recognizes conformational intact trimer

2. hc10, recognizes denatured HLA molecules

3. anti b₂M, recognizes free and non-covalently associated Beta 2microglobulin

The methodology for this assay is essentially the same as the standardElisa described above, except that instead of coating the plate withw6/32, the soluble HLA molecules are coated directly to it. The threedifferent antibodies are then utilized in three different ways to detectthe bound HLA. W6/32 is biotin labeled, therefore Vectastain kit and OPDare used for detection, anti b₂M is conjugated to horse radishperoxidase and so can be directly detected using OPD and with hc10 asecondary anti-mouse IgG horse radish peroxidase conjugated antibody andOPD are used.

Results for sHLA Production Using gDNA as the Starting Material

Using sample 3A394

Genomic DNA Extraction.

Using 200 ul whole blood, a Qiagen DNA extraction was performed. Table19 shows the optical density readings and concentration of thisextraction.

Primary PCR of 3A394

10x pfu buffer 5 ul PP5UTA 1 ul 3PPI4A 1 ul dNTP 1 ul gDNA 10 ul H₂O31.4 ul Pfu 1 ul

See FIG. 30 for the gel image of this PCR.

FIG. 30. 2% agarose gel showing the primary PCR product of 3A394 (5^(th)lane from the right) at approximately 2 kb in size.

The PCR product was gel purified and this along with unpurified PCR wasused in the secondary PCR at a 1:100 dilution.

Secondary PCR of 3A394

10x pfu buffer 5 ul dNTP 1 ul PP5UTA 1 ul PP3PEI 1 ul 1:100 1° PCR 10 ulH₂O 31.4 ul Pfu 0.6 ul

The results of this can be seen in FIG. 31. Secondary PCR of 3A394. Gelpurified and unpurified can be seen in lanes 2 and 3 of FIG. 31.

The PCR product derived from gel purified primary PCR was in turn gelpurified and used in the restriction digest in order to ligate intopcDNA 3.1(−), FIG. 32.

FIG. 32 is a gel showing 3A394 and pcDNA3.1 digested with EcoR I and XbaI. Once the ligations, at three different insert to vector ratios, andtransformation were completed colonies were picked, grown overnight andthen the plasmid DNA was extracted. A restriction digest was performedto screen for insert as shown in FIG. 33 is a restriction digests of3A394 clones. A vector to insert ratio of 1:6 is the most efficient.

Following identification of positive clones the concentration of thevector was established by optical density (Table 20) and then diluted to500 ng/ul.

Cycle sequencing was then performed on sample 3A394TPC1 and it wasestablished that this was a good clone of HLA-A*1102 truncated at codon298.

The clone 3A394PC1 was then grown up and the plasmid containing A*1102Truncated (A*1102T) was prepared for electroporation by performing alarge scale plasmid extraction. The concentration of this was determinedusing optical density which can be seen in Table 21.

30 ug of this was then electroporated into the cell line LCL 721.221with the decay times being recorded then, after 2 days the viability ofthe cells was determined, Table 22.

The cells were then placed under G418 selection to screen for positivetransfectants, those exhibiting G418 resistance were screened forsoluble HLA production using the Elisa, Table 23. Only two replicateswere tested for this sample and all were done using undilutedsupernatant.

Samples 3A394TPC1 wells 1 and 2 were grown further and eventually well 1cells were seeded into a cell pharm. This protocol has been performed onmany HLA types as can be seen by the production of soluble HLA moleculesin Table 24.

Due to the fact that the monoclonal antibody w6/32 only recognizes thefolded trimer complex of heavy chain-light chain-peptide the results ofthe ELISA demonstrate that we are producing conformational moleculesoriginally derived from genomic DNA.

FIG. 34 is a graph showing the comparative binding of three monoclonalantibodies to four different soluble HLA molecules. A*1102T is sample3A394TPC1well 1 from genomic DNA the other three are produced from cDNA.Three different amounts of soluble HLA were coated to the plate for eachallele.

The results of the comparative binding assay demonstrate severalproperties of the soluble HLA produced from genomic DNA. Coating a platewith more protein will not necessarily yield a higher signal to proteinratio. Soluble HLA from genomic DNA gives results comparable to that ofcDNA constructs. The fact that all three antibodies bind this confirmsthe correct epitopes of the recombinant molecules are present.

Due to the polymorphic nature of the HLA system production of manyalleles as soluble molecules is very difficult as a viable cell line isrequired in order to make cDNA and quite often this is not available.Thus a method that allows us to produce many different alleles from areadily available starting point is invaluable. Production of the sHLAfrom genomic DNA provides such a starting point. We have shown here thattwo simple PCR reactions allows us to clone many, if not all, HLA ClassI alleles from genomic DNA.

The Elisa data allows us to test how functional these molecule are. Byusing w6/32 and anti b₂M to establish production levels we also provideinformation as to how much of the protein is in a trimeric form. Thecomparative Elisa data helps back this up as the ratio of w6/32:hc10needs to be greater than 1.0 in order for there to be moreconformational molecule than denatured, this is shown to be the case. Insummary we have developed a technique that will allow the production ofvirtually any HLA Class I molecule in a soluble form on demand.

An exemplary useful product which can be obtained from the mammaliancell line expressing such a genomic DNA construct is a cDNA cloneencoding the desired class I or class II molecule. The cDNA cloneencoding the desired class I or class II molecule is formed from themRNA molecule encoding the desired class I molecule isolated from suchmammalian cell line. The cDNA clone may be utilized for functionaltesting, as described in more detail herein below. Thus, gDNA clones canbe used as a mechanism to obtain cDNA clones of the desired class I orclass II HLA molecule.

The cDNA clones may be transfected into a cell which is unable to spliceintrons and process the mRNA molecule and therefore would not expressthe MHC molecule encoded by the genomic DNA, such as insect cells orbacterial cells. In addition, these cell lines will also be deficient inpeptide processing and loading, and therefore the soluble MHC moleculesexpressed from such cells will not contain peptides bound therein(referred to as free heavy chain HLA). Such soluble, free heavy chainHLA can effectively be tested for epitope binding as well. That is, MHCmade in cells which do not naturally load peptide can be experimentallyloaded with the peptide of choice. The heavy chain, light chain, peptidetrimer can be reassembled in vitro using a high affinity peptide tofacilitate assembly. Alternatively, a cell deficient in peptideprocessing can be pulsed with peptide such that the trimolecular MHCcomplex forms. DNA encoding a peptide (also encoding an appropriatetargeting signal) could also be co-transfected into the cell with theMHC so that the MHC molecule which emerges from the cell is loaded onlywith the desired peptide. In this way MHC molecules could be loaded witha single low affinity peptide so that replacement with test peptides ina binding assay are more controlled.

Note that an advantage of secreting individual MHC molecules from a cellthat naturally loads peptide is that the MHC molecule of interest isnaturally loaded with thousands of different peptides. When used in apeptide binding assay, a synthetic peptide can therefore be compared tothousands of naturally loaded peptides.

Another use for the sHLA produced functional testing, according to themethodology of the present invention is the peptide-MHC complex can bemultimerized to form soluble peptide-MHC dimers or tetramers, or othermultiple soluble peptide-MHC-mers, such as fivemers, sixmers, etc. whichserve as ligands for CTLs. The tetramers can be mixed with CTLs in vitroor with CTLs from the blood of human subjects to identify antigenicpeptides responsible for immune responses in humans. Altman et al(Science, 1996), herein expressly incorporated by reference in itsentirety, discloses a method of functional testing using tetramertechnology; however, the method of Altman, however, only discloses onesoluble MHC molecule which has been utilized in such a method, andAltman's method faces the same disadvantages and defects described abovefor the prior art, that is, the method envisions isolating individualmRNA/cDNA molecules from hundreds of different, typed cell lines, andthen manipulating the cDNA molecules to produce the desired soluble MHCmolecule. The methods of the present invention envision combining thetetramer technology with amplification of genomic DNA, cloning thegenomic DNA fragment, and transfection of the resulting construct into amammalian cell line followed by isolation of cDNA from such transfectedcell line and transfection into a cell line deficient in peptideprocessing and loading, thereby removing the need to isolate hundreds ofdifferent, typed cell lines for obtaining the different cDNAs.

MHC/peptide tetramers are widely utilized in the phenotypic analysis ofT cells and in the study of T cell responses to pathological conditionssuch as viral infections and cancer. Current methodology for tetramerproduction consists of expressing the MHC class I heavy chain inbacterial or insect cells and refolding the heavy chain in the presenceof β-2-microglobulin and a specific peptide ligand in vitro.

The methodology of the present invention had two specific aims, althoughthis should not be regarded as limiting: 1) to engineer a cDNA constructof a class I heavy chain containing a BirA substrate peptide (bsp)sequence at its 3′ end (C-terminus) which would enable its subsequentbiotinylation and 2) to develop a novel means of tetramer productionusing a mammalian expression system. The mammalian system used was a Bcell/T cell hybrid, the antigen-processing mutant cell lineCEM×721.174.T2 (T2). When pulsed with established HLA B*0702-presentedpeptides from HIV-infected CD4+ T cells, T2 cells transfected with therecombinant, truncated B*0702 HLA heavy chain secreted specificMHC/peptide complexes. Following enzymatic biotinylation, thesecomplexes were combined with avidin to form B7 tetramers. A mammalianexpression system affords several advantages over a prokaryotic system,such as allowing normal glycosylation of the class I heavy chain andeliminating the need to refold the MHC/peptide complex in vitrofollowing expression. The MHC class I molecules are therefore naturallyfolded in the cell rather than artificially folded outside the cell.Indeed, such artificial folding of the MHC class I molecules outside ofthe cell results in a MHC class I molecule which differs from one foundin a “natural” system (i.e., a human immune system) and as such, is notan appropriate basis upon which to conduct vaccine development.

Abbreviations: MHC, major histocompatibility complex; β₂m,β₂-microglobulin; HLA, human leukocyte antigen; sHLA, soluble humanleukocyte antigen; bsp, BirA substrate peptide or biotinylationsubstrate peptide; CTL, cytotoxic T lymphocytes; PCR, polymerase chainreaction; ELISA, enzyme-linked immunosorbent assay; HRP, horseradishperoxidase; PE, phycoerythrin; PBS, phosphate buffered saline; TAP,Transporters associated with Antigen Processing; TCR, T cell receptor;ER, endoplasmic reticulum; kDa, kiloDaltons.

The structure and function of MHC class I. Unlike B lymphocytes whichcan interact with antigen in its intact form, T lymphocytes onlyrecognize protein antigen broken down into peptide fragments andpresented in association with specialized cell-surface molecules. Thesemolecules are the gene products of the major histocompatibility complex(MHC) (FIGS. 1 and 35), a region on chromosome six known to encodeproteins that are critical for immunologic specificity andtransplantation histocompatitiblity. MHC molecules, also termed humanleukocyte antigens (HLA), are cell-surface glycoproteins that functionprimarily in communicating to T cells the presence of intracellular orextracellular invaders. MHC/peptide complexes are recognized by the Tcell receptor (TCR), an interaction enabling T cells to discriminatebetween MHC molecules bearing “foreign” antigen (viral/tumor/bacterialpeptides) and MHC molecules displaying “self” peptides. An immuneresponse can then be initiated against cells determined to harborforeign antigen on the basis of the avidity of this binding interaction.

MHC molecules are of two types, designated class I and class II. Ingeneral, class II molecules are located on antigen presenting cells(APCs), such as macrophages or dendritic cells, and present peptidesfrom exogenously synthesized protein, usually the breakdown productsfrom phagocytosed bacteria or protozoa. Thus, class II moleculesprimarily function to warn the immune system of extracellular invaders.The class II/peptide complex is recognized by and interacts with TCRs ofCD4+ T cells (T helper or Th cells). Class I molecules are located onplatelets and all nucleated cells of the body and bind peptides derivedfrom endogenously synthesized proteins. Examples of endogenouslysynthesized proteins which may be broken up and presented by class Imolecules include both “self” proteins, such as those normally producedin healthy cells, and “foreign” proteins, such as the viral proteins invirus-infected cells or the abnormal proteins produced in tumor cells.Thus, class I molecules primarily function to report to the immunesystem the health of the intracellular environment. The TCRs of CD8+cytotoxic T lymphocytes (CTLs) interact with the MHC class I/peptidecomplex.

Structurally, class I molecules are heterodimers comprised of twononcovalently bound polypeptide chains, a larger “heavy” chain (α) and asmaller “light” chain (β-2-microglobulin, or β₂m). The polymorphic,polygenic heavy chain (45 kDa), encoded within the MHC on chromosomesix, is subdivided into three extracellular domains (designated α₁, α₂,and α₃), one intracellular domain, and one transmembrane domain (FIG.36). The two outermost extracellular domains, α₁ and α₂, together formthe groove that binds antigenic peptide (FIG. 37). Thus, interactionwith the TCR occurs at this region of the protein. The α₃ domain of themolecule contains the recognition site for the CD8 protein on the CTL;this interaction serves to stabilize the contact between the T cell andthe APC.

The invariant light chain (12 kDa), encoded outside the MHC onchromosome 15, consists of a single, extracellular polypeptide. In therecombinant truncated HLA class I molecule (soluble HLA or sHLA,approximately 47 kDa), the C-terminal intracellular and transmembranedomains of the heavy chain are not amplified by PCR and thus deleted,resulting in a functional MHC/peptide complex that is secreted from thecell and still capable of interaction with the TCR.

Antigen processing and assembly of the MHC class I/peptide complex. Theheavy chain of the class I heterodimer is cotranslationally insertedinto the lumen of the ER (FIG. 38). The extracellular (intralumenal)domains of newly synthesized a chains are glycosylated and becomeimmediately associated with the ER chaperone protein calnexin. Calnexinis a membrane-bound molecule that functions to temporarily keep theheavy chain in a partially folded state. The subsequent noncovalentinteraction of free β₂m with the heavy chain causes the release ofcalnexin, and the heavy chain/β₂m complex becomes sequentiallyassociated with the chaperones calreticulin and tapasin. Thesechaperones position the class I heterodimer in a manner that enables itto be loaded with the processed peptides once they are transported intothe ER by the TAP (transporter associated with antigen processing)complex, located within the ER membrane.

In the cytosol, a multisubunit, multicatalytic protease complex calledthe proteosome functions to prepare the endogenously synthesizedproteins for presentation by MHC class I molecules. Once proteins (viralor otherwise) are cleaved into peptides of various lengths by theproteosome, the TAP complex actively transports the peptides into the ERwhere the MHC heterodimer awaits. In the ER lumen, peptides are furthertrimmed to lengths of 8-11 amino acids. If possessing the requisitebinding affinity to the particular class I allele, a peptide will beloaded into the binding cleft of the class I heavy chain to form the MHCheavy chain/light chain/peptide heterotrimeric complex, which issubsequently routed to the cell surface via the Golgi apparatus (FIG.39).

Cells incapable of antigen processing, for reasons such as inhibition ofthe proteosome or mutation of TAP, will likewise be deficient in thecell-surface expression of the class I/peptide complex since the class Iheterodimer cannot leave the ER until it binds peptide. A particularcell line called T2, which is TAP-deficient and therefore has nocell-surface class I expression, is widely utilized in antigenprocessing studies. Additionally, it has been shown that abundantexogenous peptide can be pinocytosed, enter an intracellular traffickingpathway, and be delivered to the ER compartment directly in aTAP-independent way. High affinity peptides delivered to the ER in thismanner can then bind to de novo-synthesized MHC class I molecules and besubsequently displayed at the cell surface. Thus, T2 cells, the systemof mammalian expression utilized in the experiments herein, are capableof cell surface MHC class I expression when pulsed with exogenouspeptide of sufficient affinity for the particular class I allele.

The interaction of the MHC/peptide complex with the TCR is the centralevent in the initiation of most antigen-specific immune responses. TheTCR recognizes the antigen complex by forming intermolecular contactswith both the class I molecule and the antigenic peptide. The outcome ofthis interaction (i.e., whether or not an immune response is generated)is dependent upon the density and duration of the TCR-MHC/peptidebinding. Thus, an immunogenic MHC/peptide complex will bind the TCR withgreater avidity than a non-immunogenic MHC antigen complex. Furthermore,it is known that a monomeric MHC/peptide complex, even if immunogenic,dissociates rapidly from a TCR, indicating that multiple TCRs mustinteract with multiple MHC/peptide complexes in order to activate Tcells. In light of this information, Altman et al. disclosed a suggestedmethodology to analyze antigen-specific T cell populations bymultimerizing the MHC/peptide complex into tetramers. Essentially, atetramer consists of four biotinylated MHC/peptide complexesnon-covalently bound to an avidin molecule (FIG. 40). Compared to amonomeric MHC/peptide complex, tetramers have slower rates ofdissociation from CTLs since they are able to bind more than one TCR onthat particular CTL. This unique characteristic makes tetramers veryuseful as immunological stains.

Until now, tetramers have incorporated MHC/peptide complexes that weremade by expressing a recombinant class I heavy chain in bacteria andsubsequently refolding the heavy chain in vitro in the presence of β₂mand a specific peptide ligand. The purpose of this project was to maketetramers using MHC/peptide complexes synthesized in mammalian cells,specifically cells of the antigen-processing mutant cell line T2 so thatevery MHC/peptide complex secreted would contain the same antigenicpeptide. It is desirable to reflect as much as possible the actual invivo interaction between human cells expressing cell-surface MHC/peptidecomplexes and cytotoxic T lymphocytes.

The three following oligonucleotide primers, designated A, B, and C,were purchased from commercially available sources (i.e., operon):

Primer A, a 5′primer: (SEQ ID NO: 634)5′  GGGCCTCGAGGGACTCAGAATCTCCCCAGACGCCGAG  3′ Primer B, a 3′primer: (SEQID NO: 635) 5′  CCGCAAGCTTCCATCTCAGGGTGAGGGGCT  3′ Primer C, a 3′primer:(SEQ ID NO: 636) 5′  CCGCGAATTCTTATTCGTGCCATTCGATTTTCTG  3′

Primers A and B were used in PCR #1 and the template cDNA was arecombinant sHLA-B*0702 truncated gene with a 6-histidine tail inpcDNA3.1 (−), a mammalian expression vector. PCR #1 was designed toincorporate 5′ Xho I (CTC GAG) and 3′ Hind III (AAG CTT) cut sites andto amplify the truncated B*0702 heavy chain lacking a 3′ stop codon. AllPCR reactions for this project were performed with a proofreading taqpolymerase (PFU Polymerase, Promega) and were subjected to 25 cycles of95° C. for 1 minute (strand separation), 59° C. for 1 minute (primerannealing), and 72° C. for 2 minutes (DNA synthesis), followed by afinal extension time of 7 minutes at 72° C. The product of PCR #1 waspurified using a QIAquick PCR purification kit (QIAGEN) and wassubsequently digested for 2 hr at 37° C. with Xho I and Hind III.Concurrently, a bacterial expression vector (C-Terminal Biotin AviTagVector, Avidity; catalog number pAC-6) (FIG. 41) encoding thebiotinylation substrate peptide sequence GLNDIFEAQKIEWHE (residues 3-17of SEQ ID NO:631) was digested with the same restriction enzymes underthe same conditions. The digest products were gel purified using aFreeze N Squeeze kit (BioRad) and ligated together for 2.5 hours at roomtemperature. Competent cells of E. coli strain JM1ø9 were transformedwith the ligated DNA, plated on LB/ampicillin agar, and incubated for 16hours at 37° C. Using colony PCR, colonies were screened for insert ofthe gene into the vector.

The B*0702t-no stop/AviTag vector DNA was isolated and prepared using aDNA Miniprep kit (Promega) and served as the template cDNA for PCR #2.Primers A and C were used in PCR #2, which was designed to maintain a 5′Xho I cut site, incorporate a 3′ EcoR I cut site (GAA TTC) distal to thebsp, and amplify the B*0702t gene with the bsp on its 3′ end. The PCRproduct was purified with the QIAquick purification kit and, along withthe mammalian expression vector pcDNA3.1(−), was digested with Xho I andEcoR I restriction enzymes. Digest products were gel purified (Freeze NSqueeze), ligated together, and transformed into competent JM1ø9 E. colicells, which were then incubated overnight at 37° C. on LB/ampicillinagar. Colony PCR was performed to check selected colonies for insert ofthe B*0702t-bsp gene into pcDNA(−). DNA from clones with insert wasprepared (Miniprep kit) and sequenced using cycle sequencing. Sequenceswere analyzed and a good clone was identified. The plasmid DNA of thegood clone was isolated and prepared for transfection using a DNAMidiprep kit (QIAGEN).

Cells of the human cell line T2 were cultured in RPMI 1640 media +20%fetal calf serum, 1% penicillin/streptomycin, and 0.25% phenol red. Atotal of 1.7×10⁷ T2 cells were transfected with 30 μg ofB*0702t-bsp/pcDNA3.1 (−) DNA by electroporation using the Gene Pulser(BioRad) at 0.25 V and 960 μFD. Transfected cells were selected in amedium containing 40% RPMI 1640, 40% conditioned media, 20% fetal calfserum, 2% penicillin/streptomycin, 0.2% phenol red, and 1.5 mg/mL G418neomycin (Cellgro). Surviving cells were pulsed with the synthetic HIVGAG peptide NH₂-S-P-R-T-L-N-A-W-V-COOH (SEQ ID NO:637) at 20 ug/mL andthen incubated for 24 hours at 37° C. Transfectants were then screenedby ELISA using W6/32 (8 μg/mL), which is directed against the entire HLAheavy chain/light chain (β₂m)/peptide complex, as the primary (capture)antibody and anti-β₂m conjugated to HRP (diluted 1:1000) as thesecondary (conjugate) antibody (Dako).

High-producing cells were then expanded and cultured (continuously beingrepulsed with peptide), and their supernatant was collected andcentrifuged to remove cell debris. The specific MHC/peptide complexeswere purified using an affinity chromatography system (AKTA™ prime). Inbrief, the supernatant was passed over a 38 mL bed volume XK 26 columnof cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech)coupled to W6/32. The protein was then eluted intact in basic buffer(0.1 M glycine-NaOH, pH 11.0). To neutralize the eluate, 1.0 M Tris wasadded in a 1 to 4 ratio of neutralization buffer:elution buffer,resulting in a final solution of 0.25 M tris/0.075 M glycine, pH ˜8.0.Immediately, the eluted protein was concentrated and buffer-exchanged toPBS+0.05% Na Azide using MACROSEP centrifugal concentrators (PallFiltron, MACROSEP 10K). Purified protein was quantified using ELISA.

The purified sHLA-bsp/peptide complex was enzymatically biotinylated ona single lysine residue within the bsp by incubation with BirA (Avidity;other names for this enzyme include biotin protein ligase, biotinligase, biotin operon repressor protein, biotin holoenzyme synthetase,and biotin-[acetyl-CoA carboxylase]synthetase) (FIG. 42).

Incubation time was varied under constant conditions to determine thetime required for maximum biotinylation efficiency. Four identicalreactions were incubated for either 1, 4, 8, or 16 hours at 37° C.Biotinylation mixture components were as follows: sHLA-bsp, 6 μM; MgOAc(Biomix B, Avidity), 1.9 mM; adenosine triphosphate (Biomix B, Avidity),1.9 mM; BirA, 0.8 μM; Tris-HCl, pH 8.0, 9.4 mM; biotin (Biomix B,Avidity), 10 μM; Pefabloc SC Plus (Roche), a protease inhibitor, 0.42mM. Biotinylation was confirmed using a modified ELISA, with W6/32 (8μg/mL) as the capture antibody and ABC Vectastain (Vector Laboratories),a kit containing avidin and biotinylated HRP, as the conjugate (FIG.43).

Biotinylated sHLA was separated from free biotin by applying thebiotinylation reaction product to a Bio-Spin chromatography column(BioRad, Bio-Spin 30 Tris columns). Tetrameric sHLA/peptide complexeswere produced by the stepwise addition ( 1/10^(th) volume, waiting 10minutes between each addition) of the conjugateUltraAvidin-R-phycoerythrin (UltraAvidin-R-PE; Leinco Technologies) tothe biotinylated class I complex. The final mixture contained a 1:4molar ratio of UltraAvidin-R-PE:biotinylated class I, which is the sameas a 1:1 molar ratio UltraAvidin-R-PE biotin binding sites:biotinylatedclass I. The volumes of avidin and biotinylated class I were determinedby calculating the molar concentrations of each (in moles/μL) and thenmaking certain that the number of moles of biotin binding sites in theavidin equaled the number of moles of biotinylated class I. (Because thebiotinylation assay was qualitative rather than quantitative, theassumption that biotinylation efficiency was high (˜90%) was made priorto making these calculations.) Tetramers were purified by gel filtrationon a Superdex S-200 column (Pharmacia; Molecular Biology ResourceFacility, University of Oklahoma Health Sciences Center).

The construct successfully created using the AviTag bsp vector containeda 7-residue sequence (W-K-L-P-A-G-G) (SEQ ID NO:638) between thetruncated B7 heavy chain at its C-terminus and the bsp. Despite the factthat the peptide binding groove is at the N-terminus of the heavy chain,it was undetermined at the time of transfection whether this 7-residuelinker would in any way affect MHC protein folding or peptide bindingcapability. Because class I molecules must be properly folded and loadedwith peptide before being directed to the cell surface, only thespecific MHC/peptide complexes should be in the supernatant.Additionally, the W6/32 monoclonal capture antibody of the ELISApositively identified the presence of MHC-bsp/peptide complexes secretedinto the supernatant by producing transfectants (FIG. 44). Thus, the 7residue linker did not alter protein folding or peptide loading.

With regard to the purification of the class I-bsp molecule via affinitycolumn chromatography, the bsp addition to the molecule perhaps affectedcolumn loading, as only ˜50% recovery of the purified protein wasachieved when pre-column and post-column protein amounts were compared.A large percentage of this “lost” protein was detected both in the wash,indicating non-specific binding to the column during loading, and in theflow-thru, possibly indicating that the additional 22 amino acids (15amino acids in the bsp and 7 amino acids linking the bsp to the class I,discussed above) on the C-terminus of the molecule interfered withloading. If the latter occurred, then ELISA results quantifying sHLA-bspproduction by T2's may be underestimated, since the same W6/32 antibodyused as the capture in the ELISA is also coupled the column. The elutioncurve (FIG. 45), produced as a result of the spectrophotometric (OD280)measurements of the column system during elution, allowed determinationof which eluted fractions contained protein. These fractions were thenpooled and the purified protein was concentrated and biotinylated.

In a 1993 paper, Peter Shatz demonstrated that an array of BirAsubstrate peptide sequences, all having in common a lysine residue atposition 9 or 10, existed and were capable of being biotinylated.However, some bsp sequences biotinylate more robustly than othersequences. It is important, therefore, to determine the reaction timerequired to optimally biotinylate a given protein with its specific bsp,so as to not waste the protein in future biotinylation reactions. FIG.46 demonstrates that increasing the incubation time of the biotinylationreaction, with all other variables unchanged, allows for greaterbiotinylation.

Importantly, purifying the biotinylated class I molecules from any freebiotin remaining in the reaction mixture was performed prior to makingtetramers, as free biotin may assume one or more of the four biotinbinding sites on the avidin molecule in place of a biotinylated class Imolecule. FIG. 47 indicates that some biotinylated protein was lost inthis step. However, this purification ideally eliminates any“contamination” from monomers, dimers, or trimers when making tetramers,optimizing tetramer yield.

Upon purification of the tetramers via gel filtration (Superdex-200),tetramers (MW 498.6 kDa) were separated on the basis of size from anyother possibly-present molecule contained in the reaction mixture [freeavidin-PE conjugate (300 kDa-avidin 60 kDa and Phycoerythrin 240 kDa);free biotinylated class I (49.65 kDa); unbiotinylated class I-bsp, sincebiotinylation efficiency was not likely 100% (49.4 kDa); free biotin,despite the purification step (243 Daltons); monomers (349.65 kDa);dimers (399.3 kDa); or trimers (448.94 kDa)].

The present use of tetramers as reagents to stain T cells specific to anepitope is widely known and utilized. Additionally, the use of tetramersin vaccine development and immune modulation is a reality in presentbiomedical research. Several potential applications of tetramers inmodulating the human immune system include tolerance induction inautoimmune diseases and adoptive transfer of antigen-specific T cells inthe clinic. If the specific epitope causing the immune response in anautoimmune disease is known, a tetrameric complex specific to thepatient's HLA type and containing the immunogenic epitope could be madeand, in theory, given therapeutically to the patient in a dose largeenough to induce tolerance. Liposomes, which are artificial antigenpresenting cells that can be made and have incorporated in theirmembranes specific MHC/peptide complexes, are a cousin to the tetramerand would likely be used, rather than tetramers, for this type of immunemodulation. Adoptive transfer of T cells specific to an antigen of adisease-causing agent (tumor or viral) could be achieved by usingtetramers (specific to the patient and the antigen) to sort live Tcells, culturing these T cells in vitro to increase their numbers, andthen transferring the cells back into the patient in hopes of enhancingthe patient's immune response.

Thus, as the potential therapeutic use of tetramers in the clinicbecomes a reality, it is evident that a tetramer molecule containing MHCmolecules expressed in mammalian cells would more so reflect the proteinproduced in vivo. Although it is not known for certain the significanceof the role of MHC glycosylation in in vivo intra- and inter-molecularinteractions, it is important both in research and clinical therapy touse a system that emulates the actual interaction as much as possible.Likewise, it is not known if the class I trimeric complex formed insidemammalian cells is structurally identical to that formed outside thecell, and for this reason we choose to load sHLA class I moleculesinside the cell. In summary, we show that class I molecules can beloaded with peptide inside a mammalian cell, secreted from the cell,biotinylated, and that these biotinylated class I molecules interactwith streptavidin.

Alternatively, a cell line deficient in peptide processing but stillefficient in peptide loading may be utilized for both epitope andfunctional testing, so that a putative epitope can be expressed orpulsed into a cell and loaded into the HLA molecule in the ER of suchcell. The cDNA construct isolated as described above may be ligated intoa mammalian expression vector which also contains a DNA fragmentencoding a peptide of interest attached to a fragment encoding a signalpeptide so that the peptide of interest will be retained in the ER ofthe cell for loading, and such construct transfected into the mammaliancell line deficient in peptide processing but which retains the abilityto load peptide in the HLA molecules, such as the T2 cell line. In thismanner, the peptide of interest is produced together with the HLAmolecule. The soluble HLA molecule (with or without a His orbiotinylation signal tail) can then be purified and utilized as areagent that has been produced in mammalian cells (fully glycosylated,etc.) and is loaded with the single co-transfected peptide. Optionally,random oligomers could be made and cloned into such a mammalianexpression vector, and the soluble HLA molecules could again be purifiedand used to characterize T cells or other immune effector cells. In afurther alternative, rather than expressing the peptides with the HLAmolecule, the cells expressing the HLA molecule could be pulsed with asingle synthetic peptide or multiple synthetic peptides and analyzed asdescribed above to identify bound peptides. Any of the HLAmolecule-peptide complexes could be multimerized to form dimers,tetramers, etc. and tested for their ability to serve as ligands forCTLs and induce immune responses in humans.

In summary, the method of the present invention involves production ofMHC class I and class II molecules beginning from gDNA and/or cDNA. ThegDNA clones encoding a given MHC molecule can be truncated to besecreted rather than bound at the cell surface. This truncated versionof the MHC molecule can be produced in mammalian or insect/bacterialcells such that milligram or greater quantities of an individual class Ior class II molecule can be obtained. The secreted MHC class I moleculescan be naturally loaded with thousands of endogenous peptides inmammalian cells, while the secreted MHC class II molecules can benaturally loaded with thousands of endocytic peptides in mammaliancells. Alternatively, the secreted MHC proteins can be produced in cellsthat do not load the MHC molecule with peptide ligand. Production of MHCproteins in cells which do not load the MHC molecule with peptidefacilitates the loading of the MHC molecule via co-transfection withconstructs encoding a given peptide(s). Alternatively, the MHCpeptide-loading deficient MHC transfectant can be pulsed with peptidesor DNA encoding peptides. The resulting individual secreted MHCmolecules are useful for studies of peptide loading (i.e., in vaccinedevelopment), for characterizing human immune responses to a given MHCmolecule loaded with a particular peptide(s), and to the development ofdiagnostics where one needs sufficient MHC protein in order to directlyassess reactivity to different MHC proteins.

Another important component of the secreted MHC molecules described hereis that naturally loaded peptides can be eluted from the MHC moleculesand characterized. Substantial quantities of peptide can be obtainedfrom individual MHC molecules, and the peptides can be selectivelycharacterized. Unique information results from having a sufficientsupply of eluted peptide, and this information is essential to databasesand predictive algorithms which are essential to the vaccine architect.

Thus, in accordance with the present invention, there has been provideda methodology for producing and manipulating Class I and Class II MHCmolecules from gDNA as well as the sHLA molecules and their uses thatfully satisfies the objectives and advantages set forth herein above.Although the invention has been described in conjunction with thespecific drawings, experimentation, results and language set forthherein above, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art. Accordingly, itis intended to embrace all such alternatives, modifications andvariations that fall within the spirit and broad scope of the invention.

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TABLE 1

TABLE 2 primer type sequence (5′→3′) HLA5UT PCR (5′; inserts SalI site)GGGCGTCGACGGACTCAGAATCTCCCCAGACGCCGAG sHILA3TM PCR (3′; inserts stopcodon and HindIII site) CCGCAAGCTT

TCTCAGGGTGAG 5PXI PCR (5′; inserts XbaI site)GGGCTCTAGAGGACTCAGAATCTCCCCAGACGCCGAG 3PEI PCR (3′; inserts stop codonand EcoRI site) CCGCGAATTC

TCTCAGGGTGAG M13 universal sequencing (mp18, end through α₃)TGTAAAACGACGGCCAGT (mp19, leader through α₂) 3S sequencing (α₂ throughα₂) CGGCAAGGATTACATCGCCCTG JD3S sequencing (α₃ through end)CCCCATCGTGGGCATCGTTG 3N sequencing (α₂ through leader)CAGGGCGATGTAATCCTTGCCG 4N sequencing (α₃ through α₂)GCCAGGTCAGTGTGATCTCCGC T7 promoter sequencing (T7 promoter forwardpriming site) TAATACGACTCACTATAGGG pcDNA3.1/BGH sequencing (BGH reversepriming site) TAGAAGGCACACTCGAGG

TABLE 3 allele # fractions P2 extras P9 extras >9 cycles? B*1501 7 P —yes (14) B*1508 8 QVKRS IVMQ yes (14) B*1503 3 P MNL yes (14) B*1510 3PR MIY yes (14)

TABLE 4 allele(s) characterized ligand source protein from HLA ligandsVGYVDDTQF HLA-Iα (49-57) B*1501, 1508 IAVGYVDDTQF HLA-Iα (47-57) B*1501,B*1512 IKADHVSTY HLA-II DPα (32-40) B*1503 GSHSMRYF HLA-Iα (25-32)B*1503 Replication/transcription/translation ligands GQRKGAGSVF 60Sribosomal protein L8 B*1501, (7-16) 1503 AQAESLRY 40S ribosomal proteinS3 B*1501 (100-107) GKVRTDITY 40S ribosomal protein S4 B*1503 (73-81)SHAQTVVL 40S ribosomal protein S27 B*1510 (48-55) SQFGGGSQY eIF3-p66(61-69) B*1501, 1503, 1508, B*1512 VQGPVGTDF zinc finger transcriptionB*1501 factor (296-304) APPPPPPPP transcription factor ZFM1 B*1501(581-589) YQHTGAVL spleen mitotic checkpoint B*1510 BUB3 (53-60)AHGRKMSKSL valyl-tRNA synthetase B*1510 (859-868) LPHQPLATY Oct-bindingfactor 1 B*1508 (52-60) AKYSTPATL probable ATP-dependent B*1503 RNAhelicase DDX10 (280- 288) AKAGITTTL DNA replication licensing B*1503factor MCM5 (470-478) TQAPGNPVL splicing factor U2AF large B*1510 chain(179-187) SHQRQLLL Kin17 (49-56) B*1510 NQFQALLQY polypyrimidine tract-B*1512 binding protein (220-228) Biosynthetic/degradative modificationligands FVSNHAY aldolase A (358-364) B*1501, 1508 ILGPPGSVYubiquitin-protein ligase B*1501, (83-91) B*1502, 1508, B*1512 YMIDPSGVSYproteasome subunit C8 B*1501, (150-159) B*1502, 1508, B*4601, B*1512NHAIVSTSV 26S protease (S4) regula- B*1510 tory subunit (741-749)IHTPENPVI lanosterol 14-alpha B*1510 demethylase (488-496) AHSNLASVLO-linked GlcNAc trans- B*1510 ferase (237-245) Signalling/modulatoryligands VVAPITTGY calcyclin binding protein B*1501, (63-71) 1508GHSPPTSSL tyrosine-protein kinase B*1510 JAK3 (491-499) LPPPPPPPP Fasantigen ligand (54-62) B*1503 NHANGLTL serine/threonine protein B*1510phosphatase PP2A (α and β) (229-236) Transporter/chaperone ligandsEHVASSPAL 13S Golgi transport com- B*1510 plex 90 kD subunit (741- 749)HHSDGSVSL tapasin (354-362) B*1509, B*1510 QPGPQIVY GABA/noradrenalinetrans- B*1503 porter (261-268) Structural/eytokinesis ligands NMNDLVSEYtubulin β chain (414-422) B*1508 THTQPGVQL septin 2 homolog (70-78)B*1509, B*1510 SHANSAVVL β-adaptin (249-257) B*1509, B*1510 Unknownfunction ligands GQYPTQPTY KIAA0058 (5-13); like Mus B*1503muluscus proline-rich protein VKVIQQESY mammary tumor-associated B*1503protein INT6 (278-286) AKYPHVEDY Ki nuclear autoantigen B*1503 (207-215)AMNPTNTVF heat shock cognate 71 kD B*1503 protein (60-68) CPLSCFT humanHTGS database B*1501, B*1503, B*1508 MPHSGYGF human EST B*1508 CHSAFALhuman HTGS database B*1510 LHLLTLEA human EST B*1510 KNANLVQLY human ESTB*1512

TABLE 5 fratction ion for MA/MA derived peptide sequence 7 504.1 (+2) HM S G Z P T S Y 7 549.2 (+2) H N Z A A H Z E Y 8 526.0 (+2) H A AX Y S Z V Y 10 484.3 (+2) Y Q S D H R Y 11 424.3 (+2) HX S T Z D F 11464.3 (+2) H A P P T D P P P 11 550.0 (+2) H G P A N R D S V F 11 563.3(+2) F P Y P T D P Z Y 12 531.2 (+2) Z N A N X V Z X Y 14 585.6 (+2) R SF X X E N E Y 16 488.7 (+2) H M Z N P T S Y 16 661.9 (+2) Y V XF - - - - V Y 17 577.6 (+2) R S M X R C P E Y 18 523.0 (+2) - - F V T AZ T Y 20 582.4 (+2) M Y N C N E X D Y 25 562.8 (+2) N Q F Q A L L Q Y

TABLE 6

TABLE 7

TABLE 8 fraction ion for MS/MS derived peptide sequence 9 490.3 (+2) A GG Z P A T P P A X 9 513.1 (+2)   S H Z G C V Z P A V 10 433.8 (+2)     GH D P D S P A A 10 455.4 (+2)     E H V A S S P A L 10 482.6 (+2)     MC Z - G M P A X 10 482.8 (+2)   G H G A N N D P A X 10 495.7 (+2)   X HS Z P A G P A X 11 448.9 (+2)       M H A D N P V X 11 482.8 (+2)     GH C P R N P A X 11 495.7 (+2)   X H S G A P Z A P X 11 516.7 (+2)     XH D T E H  A  P X 12 448.4 (+2)     T Q A P G N P V L 12 460.3 (+2)    T Z A G C M  V  P X 13 464.8 (+2)       M V - - H P V X 14 456.7(+2)     A H S V P S P A F 14 477.7 (+2)     M H T - - P A P V 14 482.8(+2)     P G A A V V P S X 15 510.1 (+2)     I H T P E N P V I 16 456.7(+2)     S H D G S V P T X 16 522.7 (+2)     - - - - - - P V X 16 523.3(+2)     M A H S - - P V F 17 523.2 (+2)   - H - - - - - P V F 18 474.8(+2)     M X G X S F P A X 18 491.2 (+2)     V H T C V N P V X 18 515.8(+2)       E W H Y P V S X 19 496.6 (+2)     E T P E H A P V X

TABLE 9

TABLE 10

TABLE 11

TABLE 12

TABLE 13

TABLE 14 fraction ion for MS/MS derived peptide sequence allelesoverlapping 6 398.2 (+3) - - W D R H T X F B*1501/B*1508 6 448.2(+2) - - - - Y T B*1501/B*1508 7 418.7 (+2) A Q F A S G A G ZB*1501/B*1503 8 402.2 (+2) - G - - C D Y B*1501/B*1503 8 418.7 (+2) G SH F G V A Y B*1501/B*1508 8 516.7 (+2) N Q Z H G S A E YB*1501/B*1503/B*1508/B*1512 8 642.7 (+2) P M N D W X M T Z T YB*1501/B*1512 9 331.4 (+3) A P N A R G Z Y B*1501/B*1503 9 418.7 (+2) FV S N H A Y B*1501/B*1508 9 433.2 (+2) N P P A Z Z P N B*1501/B*1503 9437.0 (+2) T G - - - - A Y B*1501/B*1508 9 441.2 (+2) - Q - D P P P D MZ Y B*1501/B*1503 9 446.6 (+2) G Q Z Z A V D F B*1501/B*1503 9/10 465.2(+2) S Q F G G G S Q Y B*1501/B*1503/B*1508/B*1512 9 476.2 (+2) S Q F DH V T Y B*1501/B*1508 9 578.0 (+2) T P X G E P Y Z S YB*1501/B*1503/B*1508 10 398.3 (+2) X A N - - V T B*1501/B*1508 10 456.8(+2) C P L S C F T B*1501/B*1503/B*1508 10 509.0 (+2) F L Z A M Z S T YB*1501/B*1508/B*1512 10 532.0 (+2) T V X D S Z T H YB*1501/B*1508/B*1512 13 503.6 (+2) G Q R K G A G S V F B*1501/B*1503 14460.7 (+2) V V A P I T T G Y B*1501/B*1508 14 475.1 (+2) V V A C V - - -Y B*1501/B*1508 14 525.3 (+2) P L A - N - H T Y B*1501/B*1508 15 514.2(+2) F Q A R X T E Y B*1501/B*1508 16 522.0 (+2) V G Y V D D T Q FB*1501/B*1508 17 351.3 (+3) A A F C G - - X V B*1501/B*1508 17 408.7(+2) Y L H - - E T B*1501/B*1508 17/18 451.4 (+2) I L G P P G S V YB*1501/B*1508/B*1512 17 462.4 (+2) X L G D V N M Y B*1501/B*1508 17507.0 (+2) - - - - X V E F B*1501/B*1508 17 519.2 (+2) T A R V X S V E YB*1501/B*1508 18 565.7 (+2) A E F W A C Z X Y B*1501/B*1503 18/19 566.2(+2) Y M I D P S G V S Y B*1501/B*1508/B*1512 19/20 560.0 (+2) X V E X TT D Y Y B*1501/B*1512 20/21 448.2 (+2) A A G X G P T F Y B*1501/B*151220/21 614.0 (+2) I A V G Y V D D T Q F B*1501/B*1512 21/22 507.2 (+2) VA F V X F V G Y B*1501/B*1512 21/22 557.2 (+2) Y N R W  S X E FB*1501/B*1512 22/23 510.8 (+2) A L M P - - X N Y B*1501/B*1512

TABLE 15 ion overlaps positive overlap allele collided overlapsfrequency B*1512 20 14 70% B*1508 286 25 9% B*1503 88 12 14% B*1510 26 00%

TABLE 16 motif P2/P9 + length variation only + P2 variation onlyDLASMLNRY (64-72) MQLLCVF (1-7) DIEGHASHY (28-36) MLNRYKLIY (68-76)HLDIEGHASHY (26-36) SAPLEKQLF (123-131) PLEKQLFYY (125-133) MLSAPLEKQLF(121-131) APLEKQLFY (124-132) YQLRCHLSY (149-157) PLEKQLF (125-131)LPNTRPHSY (138-146) ALSINGDKF (159-167) PLEKQLFY (125-132) NTRPHSYVF(140-148) DLPDLRGPF (203-211) TMLPNTRPHSY (136-146) SINGDKFQY (161-169)FVPNLKDMF (242-250) MLPNTRPHSY (137-146) YTGAMTSKF (169-177) AVTMTAASY(253-261) QLRCHLSY (150-157) TSKFLMGTY (174-182) TMFEVSVAF (290-298)YVALSINGDKF (157-167) LTSAQSGDY (216-224) DLRWLAKSF (314-322)FQYTGAMTSKF (167-177) YSLVIVTTF (224-232) HLTTEKQEY (366-374)AMTSKFLNGTY (172-182) VIVTTFVHY (227-235) ALRLATVGY (375-383) HVLSLVF(192-198) TTFVHYANF (238-246) ALGTESGLF (467-475) SLTSAQSGDY (215-224)MTAASYARY (256-264) AVSNAVDGF (505-513) SLVIVTTF (225-232) DTETLTTMF(284-292) ALYEASTTY (564-572) LVIVTTF (226-232) ATVKGMQSY (338-346)RQIPKIQNF (597-605) IVTTFVHY (228-235) ATSVLLSAY (396-404) ILSSNYFDF(643-651) IVTTFVHYANF (228-238) SAYNRHPLF (402-410) TVMEIAGLY (666-674)FVHYANFHNF (232-241) HTVMRETLF (414-422) HVVLAIILY (679-687) FVHYANFHNFY(232-242) ESGLFSPCY (471-479) VVLAIILYF (680-688) TMTAASY (255-261)SPCYLSILRF (476-484) FLVHKIVMF (696-704) TMTAASYARY (255-264) IIPLINVTF(544-552) LVHKIVMFF (697-705) ELDTETLTTMF (282-292) TTYLSSSLF (570-578)TMFEVSVAFF (290-399) NSILSSNYF (641-649) TVLKDIIGICY (326-326) AIILYFIAF(683-691) VLKDIIGICY (327-326) FIAFALGIF (688-696) TVKGMQSY (339-346)RLATVGY (377-383) TVGYPKAGVY (380-389) LLSAYNRHPLF (400-410) PLHTVMRETLF(412-422) VMRETLF (416-422) GLALGTESGLF (465-475) GLFSPCY (473-479)LMIIPLTNVTF (542-552) PLINVTF (546-552) EVRGSALY (559-566) YLSSSLF(572-578) TQKSCIF (608-614) TQKSCIFCGF (608-617) GLETTTY (627-633)VQNSILSSNY (639-648) VQNSILSSNYF (639-649) ILSSNYF (643-649) VMEIAGLY(667-674) VVLAIILY (680-687) VVLAIILYF (680-688) VLAIILY (681-687)VLAIILYF (681-688) VLAIILYFIAF (681-691) ILYFIAF (685-691) FLVHKIVMFF(696-705)

TABLE 17 Primer name Sequence 5′-3′ Locus Cut site Annealing site PP5UTAGCGCTCTAGACCCAGACGCCGAGGATGGCC A XbaI 5UT 3PPI4A GCCCTGACCCTGCTAAAGGT AIntron4 PP5UTB GCGCTCTAGACCACCCGGACTCAGAATCTCCT B XbaI SUT 3PPI4BTGCTTTCCCTGAGAAGAGAT B Intron4 5UTB39 AGGCGAATTCCAGAGTCTCCTCAGACGCG B*39EcoRI 5UTB39 5PKCE GGGCGAATTCCCGCCGCCACCATGCGGGTCATGGCGCC C EcoRI 5UT3PPI4C TTCTGCTTTCCTGAGAAGAC C Intron4 PP5UTGGGCGAATTCGGACTCAGAATCTCCCCAGACGCCGAG B EcoRI 5UT PP3PEICCGCGAATTCTCATCTCAGGGTGAGGGGCT A, B, C EcoRI Exon 4 PP3PEIHCCGCAAGCTTTCATCTCAGGGTGAGGGGCT A, B, C HindIII Exon 4 3PEIHC7CCGCAAGCTTTCAGCTCAGGGTGAGGGGCT Cw*07 HindIII Exon 4

TABLE 18 5′CY5 Sequencing Primers Primer Name Sequence 5′-3′ T7PromTAATACGACTCACTATAGGG BGHrev TAGAAGGCACAGTCGAGG PPI2E2R GTCGTGACCTGCGCCCCPPI2E2F TTTCATTTTCAGTTTAGGCCA ABCI3E4F GGTGTCCTGTCCATTCTCA

TABLE 19 OD OD Dilution Concentration Sample 260 nm 280 nm 260 nm/280 nmfactor ug/ml 3A394 0.0346 0.0202 1.7111 20 34.5821

TABLE 20 OD OD 260 nm/ Dilution Concentration Sample 260 nm 280 nm 280nm factor ug/ml 3A394TPC1 0.2821 0.1505 1.8739 20 282.0960

TABLE 21 OD OD 260 nm/ Dilution Concentration Sample 260 nm 280 nm 280nm factor ug/ml 3A394TPC1 0.6919 0.3625 1.9087 50 1729.8492

TABLE 22 Decay time # dead Sample milliseconds # live cells/ml cells/mlViability % 3A394TPC1 19.8 1.12 × 10⁶ 1.65 × 10⁵ 87.16

TABLE 23 Optical Concentration Density of soluble Sample 492 nm DilutionHLA ng/ml 3A394TPC1 1.278 1.0 247.270 well 1 1.388 (over range)3A394TPC1 1.227 1.0 229.855 well 2 1.274 3A394TPC1 1.021 1.0 154.403well 3 1.042 3A394TPC1 1.108 1.0 169.001 well 4 1.070

TABLE 24 Concentration by ELISA ug/ml Concentration by ELISA ug/mlAllele Allele Concentration by Allele Concentration by ELISA ug/mlTotalAllele ELISA ug/ml amount made mg Total amount 545.4 3.47 made mg Totalamount made mg Total amount made mg HLA- A*0301 HLA-A*1102 888.5 2.57HLA-A*2902 476.8 2.58 HLA-A*3002 50.3 3.38 HLA-A*3201 1382.0 9.61HLA-A*3301 40.0 0.8 HLA-B*0801 66.0 21.0 HLA-B*1302 55.0 9.0 HLA-B*1401146.0 50.0 HLA-B*1801 587.6 0.4 HLA-B*3701 1831.0 119.0 HLA-B*3801 128.066.0 HLA-B*3905 1400.0 120.0 HLA-B*40012 59.0 10.0 HLA-B*4002 400.0180.0 HLA-B*4101 288.4 8.8 HLA-B*4402 59.0 10.0

TABLE A fraction ion for MS/MS derived peptide sequence 6 398.2 (+3) - -W D R H T X F 6 448.2 (+2) - - - - - Y T 7 382.7 (+2) V Q F E A A T 7418.7 ( 2) A Q F A S G A G Z 7 455.2 (+2) A L G A - - R G Y 7 489.1(+2) - - V - - G H X Y 7 506.8 (+2) X S - - - C E Y 8 402.2 (+2) - G - -C D Y 8 419.2 (+2) G S H F G V A Y 8 433.8 (+2) A P P P P P P P P 8455.2 (+2) - - - Z A R G Y 8 462.2 (+2) D P H A P P Z Y 8 507.2 (+2) A VP S X H X X Y 8 512.3 (+2) X A Z V Z M T A Y 8 512.8 (+2) A L N GR V T M Y 8 516.9 (+2) N Q Z H G S A E Y 8 522.9 (+2) F G X A C X A T SY 8 642.7 (+2) P M N D W X M T Z T Y 9 331.4 (+3) A P M A R G Z Y 9418.7 (+2) F V S N H A Y 9 426.2 (+3) - - - - - - - - S Y 9 433.3 (+2) NP P A Z Z P N 9 437.0 (+2) T G - - - - A Y 9 441.2 (+3) - Q - D P P P DM Z Y 9 446.6 (+2) G Q Z Z A V D F 9 453.6 (+2) X Q - - A G G Z Y 9465.2 (+2) S Q F G G G S Q Y 9 476.2 (+2) S Q F D H V T Y 9 481.0 (+2) GQ H A S V X S Y 9 514.2 (+2) - - A A H V P P G Y 9 550.2 (+2) F M D V GA P T V Y 9 578.0 (+2) T P X G E P Y Z S Y 10 398.3 (+2) X A N - - V T10 448.2 (+2) A Q A A P F A G Y 10 448.4 (+2) V V V F G V Z F 10 450.4(+2) A Q M - - S E Y 10 456.8 (+2) C P L S C F T 10 464.7 (+) - - - - FG H Y 10 473.7 (+2) A L W - - P Z F 10 486.4 (+2) V P H Z N A Y 10 498.7(+2) - - - - - G H G G Y 10 509.0 (+2) F L Z A M Z S T Y 10 527.7 (+2) GQ Y V V Z P T Y 10 532.0 (+2) T V X D S Z T H Y 10 540.2 (+2) P M F D PP Z T F 11 469.2 (+2) A Q A E S L R Y 11 480.6 (+2) X A V G H S G G T Y11 511.2 (+2) - - - - - P T Y 11 516.7 (+2) E S X P N N V P Y 12 383.0(+3) L A H T E C P R G Y 12 435.0 (+2) - - - - - P S Y 12 473.2 (+2)V Q G P V G V Q Y 12 475.0 (+2) R G X G V A G T A F 12 505.0 (+2) T G AP V S E E G Y 12 513.7 (+2) V Q X Y Y G S V V 12 519.0 (+2) E P A M V XZ C F 12 531.2 (+2) G Q P G A P X G G Z Y 12 541.0 (+2) GPP H N G X R AY 12 542.2 (+2) A A H W H V E A Y 12 553.7 (+2) T P P T R R E S Y 12577.2 (+2) F P T D R R S Q F 13 363.0 (+3) Y T G V S Y X H P 13 447.0(+2) A Q A S A P D A Y 13 465.0 (+2) V Q Y Y X P F 13 503.6 (+2) G Q R KG A G S V F 13 553.2 (+2) X Q Z X - - D V Y 13 590.8 (+2) A T G T A Z NX N Z Y 14 460.7 (+2) V V A P I T T G Y 14 471.5 (+2) V V A C V - - - Y14 495.2 (+2) X Q Y T V G Y F 14 525.3 (+2) P L A - N - H T Y 14 541.3(+2) P L F G Q T A G Q Y 14 550.4 (+2) A - - - - Q X E Y 14 577.2 (+2) ZG Y G N P X N G A Y 15 459.8 (+2) V Q G P V G T D F 15 470.9 (+2) V A GG W - - - F 15 514.2 (+2) F Q A R X T E Y 15 536.6 (+2) X A G F F X X EY 15 544.2 (+2) X Q - - - - Z Y 15 564.2 (+2) S G A X D R A Y Z F 16467.1 (+2) F Q - - - T X 16 500.4 (+2) T P - - - A Z A F 16 501.0 (+2) VV A T Z N Z Z X 16 503.6 (+2) Y M V T - - - F 16 517.4 (+2) A L G S Z AX M P F 16 521.3 (+2) A P A V - - - V G Y 16 522.0 (+2) V G Y V D D T QF 16 525.6 (+2) - - - - - - T G F 16 536.0 (+2) P V P N V R X N Y 16544.4 (+2) - - - - - - T X S X 16 557.6 (+2) T L E G W M S Z Y 16 561.5(+2) Y M V C N A E E Y 16 596.7 (+2) - - - - - X R D X Y 16 596.9 (+2) SL X - - - - - F 17 343.2 (+3) A Q H P S A X R F 17 351.3 (+3) A A F CG - - - X V 17 408.7 (+2) Y L H - - E T 17 441.2 (+2) - - - - - Z A Y 17451.4 (+2) I L G P P G S V Y 17 455.0 (+2) G L G Z T S A E F 17 462.4(+2) X L G D V N M Y 17 483.8 (+2) V M G X T N A N F 17 490.2 (+2) N A XG R E S S F 17 497.2 (+2) A M N P T N T V F 17 507.0 (+2) - - - - X V EF 17 511.2 (+2) X Q A P A X F V Y 17 519.2 (+2) T A R V X S V E Y 17526.8 (+2) A L E - - - E T Y 17 542.8 (+2) X Q X N A Y X S Y 17 563.2(+2) G L A R C S Z V E Y 18 503.8 (+2) S Q X A A G V D V F 18 511.7 (+2)P Q G Z M A - - Y 18 519.6 (+2) - V F V S H T T F 18 538.8 (+2) HX T GNE A T S F 18 565.7 (+2) A E F W A C Z X Y 18 566.2 (+2) Y M I T D P S GV S Y 18 581.2 (+2) X Q G H H E M F Y 20 448.2 (+2) A A G X G P T F Y 20560.0 (+2) X V E X T T D Y Y 20 614.0 (+2) I A V G Y V D D T Q F 21507.2 (+2) V A F V X F V G Y 22 510.8 (+2) A L M P - - X N Y 22 557.2(+2) Y N R W S X E F 24 546.3 (+2) - - Z D R N V T F 25 546.3 (+2) V V TM - - - Z Y * Dashes represent positions at which amino acids could notbe unambiguously assigned through NanoES-MS/MS fragmentation patternsand/or Edman data obtained. Underlined residues designate tentativeassignments.

TABLE B fraction ion for MS/MS derived peptide sequence 6 471.8 (+2) A ZV E C E T Y 7 418.7 (+2) A Q F A S G A G Z 7 504.2 (+2) Z G X G C G P AT S Y 8 402.2 (+2) - G - - C D Y 8 441.2 (+2) - - - - - Z S F 8 516.9(+2) N Q Z H G S A E Y 9 331.4 (+3) A P M A R G Z Y 9 349.4(+3) - - - - - - G F Y 9 418.7 (+2) A Z V N S G - Y 9 426.2 (+3) A A S SZ V - - P P Z Y 9 433.3 (+2) N P P A Z Z P N 9 437.0 (+2) ACG G C G Z DY 9 441.2 (+3) - Z - D P P P D M Z Y 9 446.6 (+2) G Q Z Z A V D F 9578.0 (+2) T P X G E P Y Z S Y 10 426.5 (+2) G P - - - P Z Y 10 443.2(+2) A P Z Y P P P P 10 448.3 (+2) G Z V C T P G S F 10 456.8 (+2) C P LS C F T 10 464.7 (+2) S Q F G G G S Q Y 10 465.4 (+2) A S G F N G S Z Y10 503.8 (+2) - Z - -Y T A Y 10 508.7 (+2) G Z P P H N G F Y 10 517.0(+2) I K A D H V S T Y 10 527.7 (+2) X Z A D H V X P Y 10 540.2(+2) - - - - P G Z V Y 10 549.2 (+2) Z S V - - - Z T G Y 11 437.0 (+2) HX G N Q A A Y 11 511.4 (+2) Z A G T T V P V S Y 11 527.4 (+2) G Q Y P TQ P T Y 11 581.4 (+2) F A G S Z S N T S T Y 12 494.8 (+2) S Z G G - - -T G Y 12 526.8 (+2) Z G P P N Y X T Y 12 547.1 (+2) V K V I Q Q E S Y 13454.6 (+2) L P P P P P P P P 13 476.0 (+2) A K Y S T P A T L 13 503.6(+2) G Q R K G A G S V F 13 513.1 (+2) R Z S A N H E A X 13 526.4 (+2) GK V R T D I T Y 13 553.2 (+2) V V X P A V R S T Y 13 561.0 (+2) A K Y PH V E D Y 13 571.3 (+2) A Z N X S A Y V X Y 13 601.2 (+2) E V V G D T ZY 14 438.2 (+2) A K A G I T T T L 14 490.8 (+2) V - - T Z A G S A F 14517.2 (+-2) A Z A A A N V X X Y 14 531.5 (+2) A N H S V R D T Y 14 535.3(+2) E - - - G X R Z Y 14 552.8 (+2) X Z H N D Z S T Y 14 577.2 (+2) A NE Z X G - - -Y 15 497.3 (+2) A A G P T A Z E S Y 15 514.2 (+2) V A G X VF M Z Y 15 527.0 (+2) A Z Y Z A Z V V F 15 564.2 (+2) A Z F - - - Z X Y15 577.2 (+2) Z G Y G N P X N Z Y 15 626.0 (+2) - - - - - Z A P C H Y 16521.6 (+2) A H A V Q R V V Y 16 525.6 (+2) T Z X T V V X N Y 17 446.8(+2) A Z Z A S G X A F 17 492.8 (+2) G S H S M R Y F 17 503.8 (+2) Y G YG A T V E F 17 967.6 (+1) V Z - - - T T F 18 451.4 (+2) Q P G P Q T V Y18 455.2 (+2) N G Z X S N N Y 18 475.2 (+2) A N X V Z X E Y 18 489.1(+2) G Z - - - Z G X X Y 18 497.8 (+2) A M N P T N T V F 18 525.2 (+2) YN - - - Z X F 18 538.8 (+2) - M - - S Y Z N F 18 565.7 (+2) A E F W A CZ X Y 19 521.6 (+2) S Z F G C P T R F 19 524.6 (+2) X G A X S N - - E F19 571.2 (+2) R Z A A Y R X T Y 19 646.2 (+2) T N X H D G D G A T Z Y *Dashes represent positions at which amino acids could not beunambiguously assigned through NanoES-MS/MS fragmentation patternsandlor Edman data obtained. Underlined residues designate tentativeassignments.

TABLE C fraction ion for MS/MS derived peptide sequence 6 398.2 (+3) - -W D R H T X F 6 448.2 (+2) - - - - - Y T 8 419.2 (+2) G S H F G V A Y 8441.2 (+2) V P C G Z Z S Y 8 473.2 (+2) T A Z X H R G Y 8 512.8 (+2) X AZ Y E H T Y 8 516.9 (+2) N Q Z H G S A E Y 8 546.8 (+2) N G X A M H W TY 9 418.7 (+2) F V S N H A Y 9 437.0 (+2) T G - - - - A Y 9 465.2 (+2) SQ F G G G S Q Y 9 476.2 (+2) S Q F D H V T Y 9 481.0 (+2) - P - - G Z DE V 9 514.2 (+2) N G Y D G P N A G Y 9 578.0 (+2) T P X G E P Y Z S Y 10398.3 (+2) X A N - - V T 10 448.3 (+2) M P H S G Y G F 10 450.4 (+2) V DX - - - Y 10 456.8 (+2) C P L S C F T 10 464.7 (+2) - - - - - P G F Y 10486.2 (+2) - A - P H P M G Y 10 494.2 (+2) A Q T V G Y G E Y 10 508.7(+2) - - - - - - S V Y 10 509.0 (+2) F L Z A M Z S T Y 10 532.0 (+2) T VX D S Z T H Y 11 444.1 (+2) T P - - A R A P T 11 469.2 (+2) S E H D R MY 11 480.6 (+2) T G N C S G T G T Y 11 496.8 (+2) A Q V N P S X T Y 11532.3 (+2) S P G A E T R A X Y 12 473.2(+2) Y L G - - - G A F 12 494.8(+2) X T S F M Z V Y 12 499.0 (+2) - P - - - P S S G Y 12 505.0 (+2) TP - - - G R M Y 12 513.7 (+2) P M F D Z Z V Y 12 519.0 (+2) Y L - - - RT F 12 531.2 (+2) A Q E H G C A A Z F 12 542.2 (+2) - M - - - G V H D Y12 550.2 (+2) Y V S - - R N Q Y 12 553.7 (+2) A Q Y A A G E S F Y 12564.0 (+2) T P H T Z H D E Y 12 565.2 (+2) Y M - - - F M Y 13 396.1 (+3)D P H Y V S G H Z F 13 401.2 (+2) M V G X X P A T 13 526.4 (+2) Z A S PG E X T S Y 14 460.7 (+2) V V A P I T T G Y 14 471.5 (°2) V V A CV - - - Y 14 525.3 (+2) P L A - N - H T Y 14 543.2 (+2) X A X Y R R M Y14 550.4 (+2) P L A M Z X Y T Y 15 460.6 (+2) - P - M P G X A Y 15 461.0(+2) H T T S Z N A Y 15 506.0 (+2) M A A M V G V A V Y 15 508.4 (+2) G PZ V M Z H G Y 15 514.2 (+2) F Q A R X T E Y 15 520.0 (+2) L P H Q P L AT Y 15 525.2 (+2) A A A X V - - - V T Y 15 536.6 (+2) X P E M G Z F S Y15 544.2 (+2) Y V - - V R - V F 15 564.2 (+2) F V T X N X E E Y 16 489.0(+2) A A P V G A X E S Y 16 500.4 (+2) G S - - - S Y T Y 16 522.0 (+2) VG Y V D D T Q F 16 525.7 (+2) Y V A - - - P A F 16 533.0 (+2) V G Y - -A H P G F 16 535.7 (+2) Z A T N S V T S T Y 16 537.0 (+2) - - - - - - ST Y 16 545.8 (+2) Y A T A G E M M A F 16 547.0 (+2) S P T Y T H A V A F16 557.0 (+2) M P A - - M V M A F 17 351.3 (+3) A A F C G - - - X V 17393.7 (+2) S P N E D X M Z V F 17 403.2 (+2) V A A T A G A V F 17 408.7(+2) Y L H - - E T 17 414.8 (+2) T A F P F V F 17 451.4 (+2) I L G P P GS V Y 17 462.4 (+2) X L G D V N M Y 17 476.2 (+2) Y G - - - V X S M 17490.8 (+2) X P H C S C S S F 17 504.0 (+2) D P P C W GV S F 17 507.0(+2) - - - - X V E F 17 511.2 (+2) - - - - A H D A Y 17 519.2 (+2) T A RV X S V E Y 17 526.8 (+2) X S D G R Z X T Y 17 542.8 (+2) N M N D L V SE Y 17 557.2 (+2) M P A A D Y E V A F 18 474.8 (+2) A E I L Q V I Y 18503.8 (+2) A P - - - X V S Y 18 514.7 (+2) M P A G Y N N V Y 18 519.6(+2) Y M S G X Y G T F 18 526.8 (+2) - - - A V V A Z S Y 18 538.8 (+2) XP V V P A A Z T Y 18 566.2 (+2) Y M I D P S G V S Y 18 616.3 (+2) F A NG V Z G C A F A F * Dashes represent positions at which amino acidscould not be unambiguously assigned through NanoES-MS/MS fragmentationpatterns and/or Edman data obtained. Underlined residues designatetentative assignments.

TABLE D fraction ion for MS/MS derived peptide sequence 6 493.0 (+2) N HA V G - - V S M 6 557.8 (+2) H N V F Z P T S N A 7 434.8 (+2) S V C E TE S X 7 481.3 (+2) T H P S Z A C A F 7 489.1 (+2) - H - - S P X X 8420.1 (+2) A N X E G P H T 8 441.7 (+2) G H S P P T S S L 8 494.8 (+2) CH S A F A L 8 511.6 (+2) H H A F A Z V X V 8 519.4 (+2) D H Y Y X A G SX 9 411.4 (+2) E X A P H A A X 9 424.3 (+2) A A A X R C E X 9 426.1 (+2)G H Z A P A A S X 9 441.7 (+2) V H N P Z S S X 9 444.2 (+2) A G G P T XC R X 9 455.5 (+2) L H L L T L E A 9 490.3 (+2) A G G Z P A T P P A X 9513.1 (+2) S H Z G C V Z P A V 9 520.0 (+2) X H R L C S P T X 10 404.2(+2) S V S X P H A P 10 417.1 (+2) A P F T G G N G X 10 433.8 (+2) G H DP D S P A A 10 446.2 (+2) E H G X E N G H 10 455.4 (+2) E H V A S S P AL 10 460.4 (+2) H H A P C G V S X 10 464.0 (+2) N H A I V S T S V 10464.7 (+2) G H Z N S V T S V 10 465.3 (+2) S H Z A P C T S V 10 469.4(+2) F V A R F V S X 10 469.6 (+2) H H S D G S V S L 10 473.7 (+2) S H AG A P P P T X 10 482.6 (+2) M C Z - G M P A X 10 482.8 (+2) G H G A N ND P A X 10 495.7 (+2) X H S Z P A G P A X 10 508.3 (+2) X H V V S - - VX 10 511.2 (+2) A V X D C C Z V A V 10 522.3 (+2) E X G G N T N P Z X 10522.7 (+2) Y H G S Z N P E X 10 569.6 (+2) - - - - - T Y S Y 10 574.3(+2) - - - - - - - - M 11 405.7 (+2) S H - - - Y F 11 425.8 (+2) A H P DZ A X V 11 444.7 (+2) G T A H Y Z V X 11 448.9 (+2) M H A D N P V X 11455.7 (+2) S H V D R P S X 11 459.7 (+2) T G A A F Z N P X 11 482.8 (+2)G H C P R N P A X 11 495.7 (+2) X H S G A P Z A P X 11 516.7 (+2) X H DT E H A P X 11 562.3 (+2) - - - Y Z A Y V Y 12 411.7 (+2) G H G P T X AA V 12 428.8 (+2) V P - - - - - - 12 444.7 (+2) Y Q H T G A V L 12 448.4(+2) T Q A P G N P V L 12 460.3 (+2) T Z A G C M V P X 12 490.9 (+2) T HT Q P G V Q L 12 507.4 (+2) G H A G H V P P E X 12 511.6 (+2) T H F R YV S X 12 528.1 (+2) E H R P D R V F 13 427.6 (+2) S H A Q T V V L 13449.2 (+2) S H A N S A V V L 13 464.8 (+2) M V - - H P V X 13 487.6 (+2)Y H H G G V S A F 13 506.2 (+2) - H - - G H T G Y X 14 420.1 (+2) N H AN G L T L 14 438.7 (+2) - - - - - P X X 14 456.7 (+2) A H S V P S P A F14 477.7 (+2) M H T - - P A P V 14 482.8 (+2) P G A A V V P S X 14 560.8(+2) G H A G M G C V F Z X 14 592.3 (+2) M R - - - - G X E X 15 418.9(+2) S H G V P R A X 15 439.0 (+2) E H H M P X X 15 454.3 (+2) H H Z C AA G A X 15 492.1 (+2) X V D Z A E P X V 15 510.1 (+2) I H T P E N P V I15 520.0 (+2) M G X P V R H M V 15 524.2 (+2) S H Y D W Z V X 15 532.9(+2) M P H S H P F V X 15 577.2 (+2) Z C V R C Z N G V F 16 412.9 (+2) SH A G A G X V X 16 418.3 (+2) G H X E G P X X 16 424.3 (+2) X H G G D HV X 16 448.6 (+2) E Z A H S X V X 16 448.9 (+2) Y H H D X V X 16 454.3(+2) M A G A W C R X 16 456.7 (+2) S H D G S V P T X 16 464.2 (+2) FH - - X X X 16 469.9 (+2) E H - - - T V X 16 472.3 (+2) M A X - - - - VV 16 499.0 (+2) G H A X T D G X T X 16 504.1 (+2) P V S H X V N E L 16507.7 (+2) X X Y T P G H T X 16 522.7 (+2) - - - - - - P V X 16 523.3(+2) M A H S - - P V F 16 529.9 (+2) X H Y D R N Q X 16 536.2 (+2) EA - - C Z V T T Y 16 547.9 (+2) - - - - - - A X S V 16 552.4 (+2) X ZA P T S V F Z X 17 367.7 (+3) F T M P A H P S T X 17 490.8 (+2) M T X GY G E P X 17 557.3 (+2) A H G R K M S K S L 17 340.7 (+3) - H - - H A ZV X 17 367.7 (+3) - - - - R X S H X 17 419.8 (+2) - - - H A V G X X 17462.8 (+2) M S S N E X X M 17 476.2 (+2) G H - - - - P C C 17 504.2 (+2)X H V X A V N E X 17 523.2 (+2) - H - - - - - P V F 17 543.2 (+2) X H EV Z P H X X 17 590.2 (+2) A T H C F V M E X 18 456.4 (+2) A H S N L A SV L 18 463.3 (+2) V X A P A N D X X 18 474.8 (+2) M XG X S F P A X 18491.2 (+2) V H T C V N P V X 18 497.8 (+2) S H Q R Q L L L 18 515.8 (+2)E W H Y P V S X 18 519.7 (+2) F HM D X Z T F 18 543.4 (+2) X H E V Z P HX X 18 596.8 (+2) F H H T Z S N P X X 19 434.6 (+2) - H G C P G M P X 19496.6 (+2) E T P E H A P V X 19 539.6 (+2) M X P G N S A X Y X * Dashesrepresent positions at which amino acids could not be unambiguouslyassigned through NanoES-MS/MS fragmentation patterns andlor Edman dataobtained. Underlined residues designate tentative assignments.

TABLE E fraction ion for MS/MS derived peptide sequence 7 504.1 (+2) H MS G Z P T S Y 7 549.2 (+2) H N Z A A H Z E Y 8 517.0 (+2) N Q Z H G S AE Y 8 526.0 (+2) H A A X Y S Z V Y 8 642.7 (+2) P M N D W X M T Z T Y 10465.3 (+2) S Q F G G G S Q Y 10 484.3 (+2) Y Q S D H R Y 10 509.0 (+2) FL Z A M Z S T Y 10 532.0 (+2) T V X D S Z T H Y 11 424.3 (+2) H X S T ZD F 11 464.3 (+2) H A P P T D P P P 11 550.0 (+2) H G P A N R D S V F 11563.3 (+2) F P Y P T D P Z Y 12 531.2 (+2) K N A N L V Q L Y 14 585.6(+2) R S F X X E N E Y 16 488.7 (+2) H M Z N P T S Y 16 661.9 (+2) Y V XF - - - - V Y 17 577.6 (+2) R S M X R C P E Y 18 451.1 (+2) I L G P P GS V Y 18 523.0 (+2) - - F V T A Z T Y 19 565.6 (+2) Y M I D P S G V S Y19 503.8 (+2) S Q X A A G V D V F 20 560.0 (+2) X V E X T T D Y Y 20582.4 (+2) M Y N C N E X D Y 21 448.2 (+2) A A G X G P T F Y 21 614.0(+2) I A V G Y V D D T Q F 22 507.2 (+2) V A F V X F V G Y 22 557.2 (+2)Y N R W S X E F 23 510.8 (+2) A L M P - - X N Y 25 562.8 (+2) N Q F Q AL L Q Y * Dashes represent positions at which amino acids could not beunambiguously assigned through NanoES-MS/MS fragmentation patternsand/or Edman data obtained. Underlined residues designate tentativeassignments.

1. A method for the production of soluble Class I MHC complexes in anappropriate growth media, comprising the steps of: obtaining gDNA from asample wherein a portion of the gDNA encodes a desired individual ClassI MHC heavy chain molecule; producing a PCR product encoding a solubleform of the desired Class I MHC heavy chain molecule by PCRamplification of the gDNA, wherein the amplification utilizes at leastone locus-specific primer having a stop codon incorporated into a 3′primer thereby resulting in a PCR product that does not encode thecytoplasmic and transmembrane domains of the desired Class I MHC heavychain molecule, thereby producing a PCR product that encodes a solubleClass I MHC heavy chain molecule; inserting the PCR product into amammalian expression vector to form a plasmid containing the PCR productencoding the soluble Class I MHC heavy chain molecule; electroporatingthe plasmid containing the PCR product into at least one suitable hostcell; and inoculating the appropriate growth media with the at least onesuitable host cell containing the plasmid such that soluble Class I MHCcomplexes having the desired Class I MHC heavy chain molecule associatedwith native beta-2-microglobulin and loaded with endogenously producedpeptides are produced, wherein the beta-2-microglobulin is native to andendogenously produced in the host cell.
 2. The method according to claim1, further comprising the step of harvesting the soluble Class I MHCcomplexes from the appropriate growth media.
 3. The method according toclaim 1, wherein the gDNA is obtained from blood, saliva, hair, semen,or sweat.
 4. The method according to claim 1, wherein the mammalianexpression vector contains a promoter that facilitates increasedexpression of the PCR product.
 5. The method according to claim 1,wherein the suitable host cell lacks expression of Class I MHCmolecules.
 6. A method for the production of soluble Class I MHCcomplexes in an appropriate growth media, comprising the steps of:obtaining gDNA from a sample, wherein a portion of the gDNA encodes adesired individual Class I MHC heavy chain molecule; transfecting thegDNA into a cell line, wherein the cell line transcribes the gDNA intomRNA; isolating mRNA and reverse transcribing the mRNA to obtain cDNA,wherein the cDNA contains cDNA encoding the desired Class I MHC heavychain molecule; producing a PCR product encoding a soluble form of thedesired Class I MHC heavy chain molecule by PCR amplification of thecDNA encoding the desired Class I MHC heavy chain molecule, wherein theamplification utilizes at least one locus-specific primer and results ina PCR product that does not encode the cytoplasmic and transmembranedomains of the desired Class I MHC heavy chain molecule, therebyproducing a PCR product that encodes a soluble Class I MHC heavy chainmolecule; inserting the PCR product into a mammalian expression vectorto form a plasmid containing the PCR product; electroporating theplasmid containing the PCR product into at least one suitable host cell;and inoculating the appropriate growth media with the at least onesuitable host cell containing the plasmid such that soluble Class I MHCcomplexes having the desired Class I MHC heavy chain molecule associatedwith native beta-2-microglobulin and loaded with endogenously producedpeptides are produced, wherein the beta-2-microglobulin is native to andendogenously produced in the host cell.
 7. The method according to claim6, further comprising the step of harvesting the soluble Class I MHCcomplexes from the appropriate growth media.
 8. The method according toclaim 6, wherein the gDNA is obtained from blood, saliva, hair, semen,or sweat.
 9. The method according to claim 6, wherein the locus-specificprimer includes a sequence encoding a tail such that the soluble Class IMHC heavy chain molecule encoded by the PCR product contains a tailattached thereto that facilitates in purification of the soluble Class IMHC complexes produced therefrom.
 10. The method according to claim 6,wherein the mammalian expression vector contains a promoter thatfacilitates increased expression of the PCR product.
 11. The methodaccording to claim 6, wherein the suitable host cell lacks expression ofClass I MHC molecules.
 12. The method according to claim 6, wherein thesoluble Class I MHC complexes are folded naturally and are traffickedthrough the host cell in such a way that they are identical infunctional properties to a Class I MHC complex expressed from the ClassI MHC heavy chain allele mRNA and thereby bind peptide ligands in anidentical manner as full-length, cell-surface-expressed Class I MHCcomplexes.